P2X7 inhibition of epithelial cancers and papillomas

ABSTRACT

The present invention demonstrates that P2X 7  receptor induced apoptosis may be specific for cancerous cells. Treatment with the P2X 7  ligand BzATP, increased cellular apoptosis with no associated inflammatory changes or abnormal skin or systemic effects. In mice treated with DMBA/TPA, BzATP decreased papilloma skin formation. BzATP also induced involution of developed papillomas and stimulated apoptosis in keratinocytes outgrowing at the base of developed papillomas. These data show that (a) P2X 7  regulates apoptosis of epidermal cells; (b) in vivo, local administration of a drug that activates the P2X 7  receptor can inhibit development and progression of epidermal premalignant lesions.

FIELD OF INVENTION

The present invention is related to the field of treatment andprevention of cancer. Cancer is believed to be a disease ofoverproliferation, wherein the present invention provides a method toreduce this overproliferation. For example, the methods herein result inthe induction of intracellular apoptosis, thereby resulting in theprevention of cancerous growth and/or a reduction in cancer tumor sizeand number. The administration of a specific membrane receptor agonist(i.e., for example, a P2X₇ receptor agonist) is shown herein to reducecancer growth and progression.

BACKGROUND

Cancer is a disease having many etiologies encompassing environmentaltoxins, disease, microbiological infections, and/or geneticpredispositions. As such, each causative factor can, and does, result ina different type of cancer that usually manifests in a differentbiological tissue. As a result, no one therapeutic approach has beenidentified that has been effective at slowing or preventing theprogression of a large percentage of different cancerous types. The onlycommonality that is currently recognized between all cancer diseases ismanifested by an uncontrolled cellular growth rate.

Current theories related to epithelial cell growth predicts a regulatorypathway that balances the effects of mitogenic stimuli and apoptosis.Croker et al., “Cancer stem cells: implications for the progression andtreatment of metastatic disease” J Cell Mol Med 2008, 12:374-390; andRodriguez-Nieto et al., “Role of alterations in the apoptotic machineryin sensitivity of cancer cells to treatment’ Curr Pharm Des 2006,12:4411-4425. Apoptosis is believed to be a homeostatic processorchestrated by the host's genome of selective cell deletion withoutstimulating inflammatory response. Wyllie et al., “Cell death: thesignificance of apoptosis” Int Rev Cytol 1980, 68:251-306; Ellis et al.,“Mechanisms and functions of cell death” Annul Rev Cell Biol 1991,7:663-698; and Fawthrop et al., “Mechanisms of cell death” Arch Toxicol1991, 65:437-444. Dysregulation of apoptotic cell-death has beenimplicated in states of disease and in the neoplastic transformation.Soti et al., “Apoptosis, necrosis and cellular senescence: chaperoneoccupancy as a potential switch” Aging Cell 2003, 2:39-45; and Renvoizeet al., “Apoptosis: identification of dying cells” Cell Biol Toxicol1998, 14:111-120. Present anti-cancer therapies all share a commonproblem in that normal non-cancerous cells are susceptible to thevarious treatments (i.e., for example, radiation and/or chemotherapy).

What is needed in the art is a single unified approach to cancertreatment that is directed at the common unifying mechanism ofuncontrolled growth rates. One approach having a potential for successis related to re-balancing cell proliferation/apoptosis homeostasis suchthat apoptosis predominates, such that the cell proliferation/apoptosishomeostasis is not affected in non-cancerous tissues.

SUMMARY

The present invention is related to the field of treatment andprevention of cancer. Cancer is believed to be a disease ofoverproliferation, wherein the present invention provides a method toreduce this overproliferation. For example, the methods herein result inthe induction of intracellular apoptosis, thereby resulting in theprevention of cancerous growth and/or a reduction in cancer tumor sizeand number. The administration of a specific membrane receptor agonist(i.e., for example, a P2X₇ receptor agonist) is shown herein to reducecancer growth and progression.

In one embodiment, the present invention contemplates a method fortreating cancer comprising administering a P2X₇ agonist (i.e., forexample, 3′-O-(4-benzoylbenzoyl)adenosine triphosphate; BzATP). In oneembodiment, the P2X₇ agonist activates apoptosis. In one embodiment, thecancer comprises a papilloma. In one embodiment, the cancer comprises anepithelial cancer. In one embodiment, the apoptosis is activated in thepapilloma. In one embodiment, the apoptosis is activated in theepithelial cancer. In one embodiment, the apoptosis is not activated innon-cancerous tissue.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a subject comprising a plurality ofmalignant cancer cells and a plurality of non-cancer cells; ii) a P2X₇receptor agonist capable of inducing apoptosis in said cancer cells; b)administering said agonist to said cancer cells under conditions suchthat apoptosis is induced. In one embodiment, the agonist comprisesBzATP. In one embodiment, the apoptosis kills the cancer cell. In oneembodiment, the agonist does not induce apoptosis in the non-cancerouscells. In one embodiment, The administering is selected from the groupconsisting of intradermal, intratumoral, transdermal, intraperitoneal,intranasal, intravenous, intramuscular, and subcutaneous. In oneembodiment, the administering further comprises TNFα. In one embodiment,the cancer cell is derived from an epithelial cell. In one embodiment,the cancer cell comprises a squamous cell carcinoma. In one embodiment,the cancer cell comprises a P2X₇ receptor. In one embodiment, thenon-cancer cell comprises a P2X₇ receptor.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a subject at risk for developing aplurality of benign cancer cells; ii) a P2X₇ receptor agonist capable ofinducing apoptosis in said cancer cells; b) administering said agonistto said subject under conditions such that apoptosis is induced, therebypreventing the development of said cancer cells. In one embodiment, theagonist comprises BzATP. In one embodiment, the apoptosis kills saiddeveloping cancer cell. In one embodiment, the administering is selectedfrom the group consisting of intradermal, intratumoral, transdermal,intraperitoneal, intranasal, intravenous, intramuscular, andsubcutaneous. In one embodiment, the administering further comprisesTNFα. In one embodiment, the developing cancer cell is derived from anepithelial cell. In one embodiment, the developing cancer cell comprisesa papilloma. In one embodiment, the subject further comprises aplurality of non-cancerous cells. In one embodiment, the agonist doesnot induce apoptosis in the non-cancerous cells. In one embodiment, thecancer cell comprises a P2X₇ receptor. In one embodiment, thenon-cancerous cell comprises a P2X₇ receptor.

In one embodiment, the present invention contemplates a method oftreating and/or preventing cancer by administering compounds capable ofbinding to a P2X₇ receptor gene enhancer region, wherein the enhancerregion increases P2X₇ receptor expression. In one embodiment, the P2X₇gene comprises an enhancer region (i.e., for example, +222/+232). In oneembodiment, the compounds modulate P2X₇ receptor expression regulatorproteins. In one embodiment, the regulator proteins may be selected fromthe group including, but not limited to, p300, Elk-1, E47, E11aE, E2F,or p53. In one embodiment, a sequence of the putative enhancer region+222/+232 comprises binding sites for regulator proteins selected fromthe group including, but not limited to, p300, Elk-1, E47, E11aE, E2F,or p53.

DEFINITIONS

The term “cancer”, as used herein refers to any presence of cellspossessing characteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells, for example, leukemia cells. The number of cancercells in a subject's body can be determined by direct measurement, or byestimation from the size of primary or metastatic tumor masses. Forexample, the number of cancer cells in a subject can be measured byimmunohistological methods, flow cytometry, or other techniques designedto detect characteristic surface markers of cancer cells.

The term “inhibit the proliferation of a cancer cell” as used herein,means to kill a cancer cell, or permanently or temporarily arrest orslow the growth of a cancer cell. Inhibition of cancer cellproliferation can be inferred if the number of cancer cells in a subjectremains constant or decreases after administration of a miRNA geneproduct and/or an miRNA gene expression-inhibiting compound. Aninhibition of cancer cell proliferation can also be inferred if theabsolute number of such cells increases, but the rate of tumor growthdecreases.

The term “detecting” as used herein, refers to obtaining indirectevidence regarding the likelihood of the presence of apathophysiological condition or assessing the predisposition of anorganism to the development of the pathophysiological condition (i.e.,for example, epithelial cancer).

The term “diagnosing” as used herein, refers to establishing scientificevidence demonstrating the actual presence of a pathophysiologicalcondition (i.e., for example, epithelial cancer).

The term “tumor” or “papilloma” as used herein, refers to all neoplasticcell growth and proliferation, whether malignant or benign, and allprecancerous and cancerous cells and tissues. The size of a tumor can beascertained by direct visual observation, or by diagnostic imagingmethods, including, but not limited to, X-ray, magnetic resonanceimaging, ultrasound, and scintigraphy. Diagnostic imaging methods usedto ascertain size of the tumor can be employed with or without contrastagents. The size of a tumor can also be ascertained by physical means,such as palpation of the tissue mass or measurement of the tissue masswith a measuring instrument, such as a caliper.

The term “precancerous” as used herein, refers to cells or tissueshaving characteristics relating to changes that may lead to malignancyor cancer. Examples include, but are not limited to, adenomatous growthsin uterus, skin, colon, ovary, breast, or prostate. Examples alsoinclude, abnormal neoplastic, in addition to dysplastic nevus syndromes,polyposis syndromes, prostatic dysplasia, and other such neoplasms,whether the precancerous lesions are clinically identifiable or not.

The term “tumor-cell killing” as used herein, refers to any inhibitionof tumor cell proliferation by means of blocking a function or bindingto block a pathway related to tumor-cell proliferation. For example,inhibition of an mRNA (i.e., for example, P2X₇ mRNA) may block a pathwayresulting in tumor-cell proliferation.

The term “anti-P2X₇ antibody” as used herein, refers to any antibody orantibody fragment that specifically binds a polypeptide encoded by aP2X₇ gene, mRNA, cDNA, or a subsequence thereof. These antibodies canmediate anti-proliferative activity on tumor-cell growth.

The term “immunoassay” as used herein, refers to any assay that utilizesthe binding interaction between an antibody and an antigen. Typically,an immunoassay uses the specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

The term “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with” as used herein, whenreferring to a protein or peptide, refers to any binding reaction thatis determinative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. For example, underdesignated immunoassay conditions, specified antibodies bind to aparticular protein at a level at least two times the background and donot substantially bind in a significant amount to other proteins presentin the sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, antibodies raised to a particular P2X₇polypeptide can be selected to obtain only those antibodies that arespecifically immunoreactive with a P2X₇ polypeptide, and not with otherproteins, except for polymorphic variants, orthologs, and alleles of aspecific P2X₇ polypeptide.

The term “cancer symptoms” as used herein, refers to observable changesin a subject's physical and/or medical condition consistent with aspecific type of cancer. In general, cancer symptoms may include, butare not limited to, weight loss, fatigue, localized swelling, orlocalized pain. Each cancer type comprises symptoms that may or may notoccur in a different type of cancer. For example, symptoms of uterinecancer include, but are not limited to, abnormal bleeding, spotting, orother discharges from the vagina. On the other hand, symptoms ofcervical cancer include, but are not limited to, continuous vaginaldischarge, abnormal and/or heavy vaginal bleeding, loss of appetite,pelvic and/or back pain, single swollen leg, or bone fractures.

The term “suspected” or “suspecting” as used herein, refers to a medicaldeduction based upon the observance of cancer symptoms concluding that asubject (i.e., for example, a patient and/or mammal) may have contracteda disease or other medical condition (i.e., for example, cancer).

The term “in need of a diagnosis” as used herein, refers to a patient orsubject (i.e., for example, a mammal) comprising at least onehypermethylated gene region (i.e., for example, a hypermethylated P2X₇gene region).

The term “in need of a treatment” as used herein, refers to a patient orsubject (i.e., for example, a mammal) having been diagnosed with adisease or other medical condition (i.e., for example, cancer).

The term “local” as used herein, refers to the non-parenteraladministration of a therapeutic agent. A local administration mayinclude, but is not limited to topical or intratumoral. A minimal amountof systemic distribution is expected during a local administration butwould be expected to maintain subclinical thresholds.

The term “detecting” as used herein, refers to obtaining indirectevidence regarding the likelihood of the presence of a disease and/orassessing the predisposition of an organism to the development of thedisease (i.e., for example, uterine cancer).

The term “diagnosing” as used herein, refers to establishing scientificevidence demonstrating the actual presence of a disease (i.e., forexample, uterine cancer).

The term “tumor” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues. The size of a tumor can be ascertained bydirect visual observation, or by diagnostic imaging methods, including,but not limited to, X-ray, magnetic resonance imaging, ultrasound, andscintigraphy. Diagnostic imaging methods used to ascertain size of thetumor can be employed with or without contrast agents. The size of atumor can also be ascertained by physical means, such as palpation ofthe tissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The term “precancerous” as used herein, refers to cells or tissueshaving characteristics relating to changes that may lead to malignancyor cancer. Examples include, but are not limited to, adenomatous growthsin uterus, skin, colon, ovary, breast, or prostate. Examples alsoinclude, abnormal neoplastic, in addition to dysplastic nevus syndromes,polyposis syndromes, prostatic dysplasia, and other such neoplasms,whether the precancerous lesions are clinically identifiable or not.

The term “at risk for” as used herein, refers to a medical condition orset of medical conditions exhibited by a patient which may predisposethe patient to a particular disease or affliction. For example, theseconditions may result from influences that include, but are not limitedto, behavioral, emotional, chemical, biochemical, or environmentalinfluences.

The term “effective amount” as used herein, refers to a particularamount of a pharmaceutical composition comprising a therapeutic agentthat achieves a clinically beneficial result (i.e., for example, areduction of symptoms). Toxicity and therapeutic efficacy of suchcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,”“prevent” and grammatical equivalents (including “lower,” “smaller,”etc.) when in reference to the expression of any symptom in an untreatedsubject relative to a treated subject, mean that the quantity and/ormagnitude of the symptoms in the treated subject is lower than in theuntreated subject by any amount that is recognized as clinicallyrelevant by any medically trained personnel. In one embodiment, thequantity and/or magnitude of the symptoms in the treated subject is atleast 10% lower than, at least 25% lower than, at least 50% lower than,at least 75% lower than, and/or at least 90% lower than the quantityand/or magnitude of the symptoms in the untreated subject.

The term “inhibitory compound” as used herein, refers to any compoundcapable of interacting with (i.e., for example, attaching, binding etc)to a binding partner under conditions such that the binding partnerbecomes unresponsive to its natural ligands Inhibitory compounds mayinclude, but are not limited to, small organic molecules, antibodies,and proteins/peptides.

The term “attached” as used herein, refers to any interaction between amedium (or carrier) and a drug. Attachment may be reversible orirreversible. Such attachment includes, but is not limited to, covalentbonding, ionic bonding, Van der Waals forces or friction, and the like.A drug is attached to a medium (or carrier) if it is impregnated,incorporated, coated, in suspension with, in solution with, mixed with,etc.

The term “medium” as used herein, refers to any material, or combinationof materials, which serve as a carrier or vehicle for delivering of adrug to a treatment point (e.g., wound, surgical site etc.). For allpractical purposes, therefore, the term “medium” is consideredsynonymous with the term “carrier”. It should be recognized by thosehaving skill in the art that a medium comprises a carrier, wherein saidcarrier is attached to a drug or drug and said medium facilitatesdelivery of said carrier to a treatment point. Further, a carrier maycomprise an attached drug wherein said carrier facilitates delivery ofsaid drug to a treatment point. Preferably, a medium is selected fromthe group including, but not limited to, foams, gels (including, but notlimited to, hydrogels), xerogels, microparticles (i.e., microspheres,liposomes, microcapsules etc.), bioadhesives, or liquids. Specificallycontemplated by the present invention is a medium comprisingcombinations of microparticles with hydrogels, bioadhesives, foams orliquids. Preferably, hydrogels, bioadhesives and foams comprise any one,or a combination of, polymers contemplated herein. Any mediumcontemplated by this invention may comprise a controlled releaseformulation. For example, in some cases a medium constitutes a drugdelivery system that provides a controlled and sustained release ofdrugs over a period of time lasting approximately from 1 day to 6months.

The term “drug” or “compound” as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars.

The term “administered” or “administering” a drug or compound, as usedherein, refers to any method of providing a drug or compound to apatient such that the drug or compound has its intended effect on thepatient. For example, one method of administering is by an indirectmechanism using a medical device such as, but not limited to a catheter,applicator gun, syringe etc. A second exemplary method of administeringis by a direct mechanism such as, local tissue administration (i.e., forexample, extravascular placement), oral ingestion, transdermal patch,topical, inhalation, suppository etc.

The term “patient”, as used herein, is a human or animal and need not behospitalized. For example, out-patients, persons in nursing homes are“patients.” A patient may comprise any age of a human or non-humananimal and therefore includes both adult and juveniles (i.e., children).It is not intended that the term “patient” connote a need for medicaltreatment, therefore, a patient may voluntarily or involuntarily be partof experimentation whether clinical or in support of basic sciencestudies.

The term “affinity” as used herein, refers to any attractive forcebetween substances or particles that causes them to enter into andremain in chemical combination. For example, an inhibitor compound thathas a high affinity for a receptor will provide greater efficacy inpreventing the receptor from interacting with its natural ligands, thanan inhibitor with a low affinity.

The term “derived from” as used herein, refers to the source of acompound or sequence. In one respect, a compound or sequence may bederived from an organism or particular species. In another respect, acompound or sequence may be derived from a larger complex or sequence.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general,a protein comprises amino acids having an order of magnitude within thehundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term “pharmaceutically” or “pharmacologically acceptable”, as usedherein, refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human.

The term, “pharmaceutically acceptable carrier”, as used herein,includes any and all solvents, or a dispersion medium including, but notlimited to, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, coatings, isotonic and absorption delayingagents, liposome, commercially available cleansers, and the like.Supplementary bioactive ingredients also can be incorporated into suchcarriers.

The term, “purified” or “isolated”, as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the composition(i.e., for example, weight/weight and/or weight/volume). The term“purified to homogeneity” is used to include compositions that have beenpurified to ‘apparent homogeneity” such that there is single proteinspecies (i.e., for example, based upon SDS-PAGE or HPLC analysis). Apurified composition is not intended to mean that some trace impuritiesmay remain.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

The term “biocompatible”, as used herein, refers to any material doesnot elicit a substantial detrimental response in the host. There isalways concern, when a foreign object is introduced into a living body,that the object will induce an immune reaction, such as an inflammatoryresponse that will have negative effects on the host. In the context ofthis invention, biocompatibility is evaluated according to theapplication for which it was designed: for example; a bandage isregarded as biocompatible with the skin, whereas an implanted medicaldevice is regarded as biocompatible with the internal tissues of thebody. Preferably, biocompatible materials include, but are not limitedto, biodegradable and biostable materials.

The term “biodegradable” as used herein, refers to any material that canbe acted upon biochemically by living cells or organisms, or processesthereof, including water, and broken down into lower molecular weightproducts such that the molecular structure has been altered.

The term “bioerodible” as used herein, refers to any material that ismechanically worn away from a surface to which it is attached withoutgenerating any long term inflammatory effects such that the molecularstructure has not been altered. In one sense, bioerosin represents thefinal stages of “biodegradation” wherein stable low molecular weightproducts undergo a final dissolution.

The term “bioresorbable” as used herein, refers to any material that isassimilated into or across bodily tissues. The bioresorption process mayutilize both biodegradation and/or bioerosin.

The term “biostable” as used herein, refers to any material that remainswithin a physiological environment for an intended duration resulting ina medically beneficial effect.

“Nucleic acid sequence” and “nucleotide sequence” as used herein referto an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.

The term “an isolated nucleic acid”, as used herein, refers to anynucleic acid molecule that has been removed from its natural state(e.g., removed from a cell and is, in a preferred embodiment, free ofother genomic nucleic acid).

The terms “amino acid sequence” and “polypeptide sequence” as usedherein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

The term “antibody” refers to immunoglobulin evoked in animals by animmunogen (antigen). It is desired that the antibody demonstratesspecificity to epitopes contained in the immunogen. The term “polyclonalantibody” refers to immunoglobulin produced from more than a singleclone of plasma cells; in contrast “monoclonal antibody” refers toimmunoglobulin produced from a single clone of plasma cells.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., for example, an antigenic determinant orepitope) on a protein; in other words an antibody is recognizing andbinding to a specific protein structure rather than to proteins ingeneral. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabelled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

As used herein, the term “antisense” is used in reference to RNAsequences which are complementary to a specific RNA sequence (e.g.,mRNA). Antisense RNA may be produced by any method, including synthesisby splicing the gene(s) of interest in a reverse orientation to a viralpromoter which permits the synthesis of a coding strand. Once introducedinto a cell, this transcribed strand combines with natural mRNA producedby the cell to form duplexes. These duplexes then block either thefurther transcription of the mRNA or its translation. In this manner,mutant phenotypes may be generated. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense” strand. The designation (−) (i.e., “negative”) is sometimes usedin reference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The term “a cell comprising a P2X₇ receptor” as used herein, refers toany cell derived from a bodily tissue displaying a P2X₇ receptor.wherein activation of the receptor induces apoptosis. For example, suchcell include, but are not limited to, epithelial cells, neuronal cells,glial cells, endothelial cells, bone marrow cells, muscle cells,hemopoietic cells, white blood cells, gastrointestinal cells, urinarytract cells, gonadal cells, renal cells, pancreatic cells, retinalcells, prostate cells, lung cells, or kidney cells.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 presents exemplary data showing P2X₇ expression in epidermalcells. Panels a-d are representative of 2-6 repeats.

Micrographs a and b: P2X₇ mRNA in-situ hybridization of human normalkeratinocytes; a anti-sense probe, b sense probe (×10).

Micrographs c and d (×40): P2X₇ immunostains of cultured human normalkeratinocytes (c) and SCC-9 cells (d); arrows point to cells membranes.In c and d the immunoreactivity to the anti-P2X₇ antibody was blocked bypre-incubation with the P2X₇ antigen (not shown).

Micrograph e: Quantitative analysis of P2X₇ immunostaining (empty bars)and P2X₇ mRNA qPCR (normalized to E-Cadherin mRNA, filled bars) in humannormal keratinocytes (K) and SCC-9 (S-9) cells (means±SD, n=3);*-p<0.01.

Insert in e: Effects of treatments with ATP on apoptosis. Human normalkeratinocytes (K) and SCC-9 (S-9) cells were treated with 250 μM ATP for8 hrs; Control—no treatment. Shown are means±SD of 3 experiments. * and**-p<0.03-0.01 compared to k; ***-p<0.01 compared to control.AU—arbitrary units.

Micrographs f and g: Cross-sections (×10) of mouse dorsal skin; f-P2X₇immunostain, g-phase. The vertical arrow in f points the epidermis andthe horizontal arrow points a hair shaft.

FIG. 2A presents exemplary data showing apoptosis dose-related effectsof ATP and BzATP in human normal keratinocytes (means of 3 experiments,variability ranged 3-8%).

Insert a: Effects on apoptosis of treatments with ATP, BzATP, and TNFα,alone or in combination (means±SD, n=3-5). Data were normalized toapoptosis level (=1) in non-treated cells (Baseline condition).*-p<0.01.

Insert b: Steady-state levels of ATP in conditioned media of culturedhuman normal keratinocytes (K) and SCC-9 (S-9) cells (means±SD, n=3).

FIGS. 2B and 2C present exemplary data showing heterologous expressionin MDCK cells of the human full-length P2X₇ cDNA (Myc-tagged at theN-terminus), and effects of treatments on baseline and of BzATP-inducedapoptosis (100 μM, 8 hrs). Cells were also co-treated with the vehicle(Control); with anti-sense P2X₇ oligonucleotides (ASO), or withrandom-control P2X₇ oligonucleotides (RCO) (means±SD, n=3).WT—wild-type.

Insert: P2X₇ immunoreactivity in N-Myc-hP2X7-MDCK cells captured bylaser confocal microscopy at the planar (a) and vertical (b) axes.

FIGS. 2C-2E present exemplary data showing Western immunoblots oflysates obtained from N-Myc-hP2X7-MDCK cells treated with therandom-control P2X₇ oligonucleotides (RCO) (c) or with the anti-senseP2X₇ oligonucleotides (ASO) (d and e) (n=3). The c and d lanes representgel blotted initially with the anti Myc antibody and reprobed with theanti P2X₇ antibody. Experiments were repeated 3 times with similarresults. * and **-p<0.01 compared to WT-MDCK. AU—arbitrary units.

FIG. 3 presents exemplary data showing BzATP effects in vivo onepidermal apoptosis.

FIGS. 3A-F: Experiment 1: Cross-sections (×40) taken from the anterior(Control) and posterior (BzATP-treated) regions of the dorsal skin ofthe same mouse. Sections were immunostained with anti-P2X₇ antibody andco-stained with DAPI for nuclear morphology. Arrows in d point to nucleiof P2X₇-positive epidermal cells in basal/parabasal regions of theepidermis at stages of condensation, fragmentation and pyknosis.

Figures G-L: Experiment-2: Cross-sections (×10) were either stained withH&E (g,h), immunostained with anti-Ki67 antibody (i,j), or stained forTUNEL (apoptosis) (k,l). Arrows in 1 point to increased TUNEL stainingin BzATP-treated cells in basal/parabasal layers of the epidermis(horizontal arrow) and in a hair shaft (vertical arrow).

Figures M-X: Experiment-2: Cross-sections (m-r, x10; s-x x20)immunostained with anti-P2X₇ antibody and co-stained for TUNEL(apoptosis). Arrows in r show co-localization of P2X₇ immunoreactivitywith TUNEL in epidermal cells in the basal/parabasal layers (horizontalarrow) and in a hair shaft (vertical arrow) of BzATP-treated animals.Experiments were repeated 3-5 times with similar results. Panel x showsenhanced TUNEL staining co-localized with P2X₇ immunoreactivity inepidermal cells from BzATP-treated animal in the basal/parabasal layersand in a hair shaft. Experiments were repeated 3-5 times with similarresults.

FIG. 4 present exemplary data showing BzATP effects on skin papillomaformation in DMBA/TPA-treated mice. Treatments are described herein asExperiment-3. Shown are means (±SEM) of data obtained in the DMBA/TPAgroup (filled circles, n=15) and in the DMBA/TPA+BzATP group (emptycircles, n=14).

FIG. 4A: Percent animals with at least one papilloma.

FIG. 4B: Number of papillomas per animal.

FIG. 4C: Mean papilloma size (mm [largest lesion dimension]).

FIG. 5 presents illustrative photographs of gross morphology of skinpapillomas in DMBA/TPA− and in DMBA/TPA+BzATP-treated mice followingtreatments described herein as Experiment-3. Arrows in c point topapillomas at various stages of involution. FIGS. 5A and 5B representhematoxylin/eosin (H&E) staining. FIGS. 5C and 5B represent TUNELstaining (c,d) (×10). Arrow in d points to increased TUNEL staining inbasal/parabasal layers of outgrowing keratinocytes in the papilloma.

FIG. 6 presents representative photographs of DMBA/TPA-induced skinlesions in mice in-vivo, and the effects of co-treatment with BzATP.Arrows in D and E point to involuting papillomas. Treatments aredescribed herein as Experiment-4.

FIG. 7 presents representative histological cross-sections, evaluatedhistologically by H&E staining, of DMBA/TPA-induced skin lesions in micein-vivo, and the effects of co-treatment with BzATP.

FIG. 8 presents exemplary data showing a summary of the effects of localtreatments with DMBA/TPA (black symbols) or DMBA/TPA+BzATP (whitesymbols) on the proportion of living mice with skin lesions. Treatmentis described herein as Experiment-4. (expressed as mean data; standarddeviation (SD) ranges between 3-11%).

FIG. 8A: Skin lesions at 0-12 weeks of treatment were papillomas. Skinlesions at 14-28 weeks of treatment were grouped either as cancerouslesions (squamous spindle-cell carcinomas, circles), or as non-cancerouslesions (existing or involuting papillomas, triangles).

FIG. 8B: Skin lesions at 14-28 weeks of treatment were grouped either ascancerous lesions (squamous spindle-cell carcinomas, circles), or asnon-cancerous lesions (existing or involuting papillomas, triangles).

FIGS. 9A-9C present exemplary data showing a summary of the effects oflocal treatments with DMBA/TPA (black symbols) or DMBA/TPA+BzATP (whitesymbols) on the mean number of skin lesions per living animal.Treatments are described herein as Experiment-4. Values represent means,and standard deviations ranged between 5-9%.

FIG. 10 presents exemplary data showing a summary of the effects oflocal treatments with DMBA/TPA (black symbols) or DMBA/TPA+BzATP (whitesymbols). Treatments are described herein as Experiment-4. Valuesrepresent means, and standard deviations ranged between ranged 2-18%.

FIG. 10A: Mean lesion size between 0-12 weeks.

FIG. 10B: Proportion of living mice with total lesions volume per animalof >10 mm³

FIG. 10C: Proportion of living mice with total lesions volume per animalof >200 mm³.

FIG. 11 presents exemplary data showing a summary of the effects oflocal treatments with DMBA/TPA (black symbols) or DMBA/TPA+BzATP (whitesymbols). Treatments are described herein as Experiment-4.

FIG. 11A: Time-to-event of cancer state.

FIG. 11B: Survival rates.

FIG. 12 presents exemplary data showing P2X₇ immunoreactivity in mice.Treatments are described herein as Experiment-4.

FIG. 12A: Normal epidermis.

FIG. 12B: An H&E stained parallel cross section of a normal epidermis.

FIG. 12C. Papilloma epidermis.

FIG. 12D: An H&E stained parallel cross section of a papillomaepidermis.

FIG. 12E: Cancerous epidermis.

FIG. 12F: An H&E stained parallel cross section of a cancerousepidermis.

FIG. 12G: Analysis of P2X₇ immunoreactivity compared among pairedhistologically normal and cancerous tissues. Bars are means (±SD) oflevels in tissues of five mice. Normal tissue data was normalized ineach case to an arbitrary value of 10. *-p<0.01. AU—arbitrary units.

FIG. 12H: P2X₇ protein assays in mouse normal and cancer skin tissues.Lysates fractionated by gel electrophoresis were immunoblotted with theanti-P2X₇ antibody and membranes were re-probed with the anti-GAPDHantibody. Normal tissue data was normalized in each case to an arbitraryvalue of 10. *-p<0.01. AU—arbitrary units.

Insert: Western immunoblot of lysates of histologically normal andcancerous tissues obtained from the same animal. Similar results wereobtained in tissues of two additional mice. Bars show means (±SD) ofdensitometry results of the P2X₇-specific 75 KDa bands in tissues ofthree mice.

FIG. 12I: P2X₇ mRNA levels (relative to GAPDH mRNA) (means±SD) inhistologically normal and cancerous tissues obtained from three animals.Normal tissue data was normalized in each case to an arbitrary value of10. *-p<0.01. AU—arbitrary units.

FIG. 13 presents exemplary data showing the effects in mice (n=5)in-vivo of local treatment with BzATP on skin apoptosis. Treatments aredescribed herein as Experiment-5. BzATP (B) was applied on the shavedanterior skin area with the Control (C) applied in parallel on theshaved posterior skin area. The horizontal line shows schematicallyseparation of the anterior and posterior dorsal skin regions. Data inA-I are representative of similar results in five animals.

FIG. 13A: Gross morphology of an untreated control mouse.

FIG. 13B: An H&E stained cross section of BzATP treated anterior skin.

FIG. 13C: An H&E stained cross section of control posterior skin.

FIG. 13D: A TUNEL stained cross section of control posterior skin in theabsence of TdT.

FIG. 13E: A TUNEL stained cross section of BzATP treated anterior skinin the absence of TdT.

FIG. 13F: A TUNEL stained cross section of control posterior skin in thepresence of TdT.

FIG. 13G: A TUNEL stained cross section of BzATP treated anterior skinin the presence of TdT. Arrows: Enhanced TUNEL staining in epidermalcells of the basal/parabasal layers (horizontal arrow) and of epidermalhair shaft cells (vertical arrow).

FIG. 13H: Nuclear staining with DAPI in control posterior skin.

FIG. 13I: Nuclear staining with DAPI in BzATP treated anterior skin.Arrows: Nuclei of epidermal cells in basal/parabasal regions of theepidermis at advanced stages of condensation, fragmentation andpyknosis.

FIG. 14 presents exemplary data showing the effects in mice in-vivo oflocal treatment with BzATP on skin apoptosis. Treatments are describedherein as Experiment-6. Animals (n=5) were treated for 16 weeks eitherwith BzATP, applied locally twice a week. Assays were repeated 3-5 timeswith similar trends

FIG. 14A: Gross morphology of dorsal skin treated with BzATP.

FIG. 14B: Cross section of dorsal skin treated with BzATP with H&Estain.

FIG. 14C: Gross morphology of dorsal skin treated with vehicle.

FIG. 14D: Cross section of dorsal skin treated with vehicle with H&Estain.

FIG. 14E: Cross section of dorsal skin treated with vehicle with P2X₇immunostain.

FIG. 14F: Cross section of dorsal skin treated with BzATP with P2X₇immunostain.

FIG. 14G: Cross section of dorsal skin treated with vehicle with TUNELstain.

FIG. 14H: Cross section of dorsal skin treated with BzATP with TUNELstain.

FIG. 14I: Overlay of FIG. 14E and FIG. 14G showing co-localization atlow magnification.

FIG. 14J: Overlay of FIG. 14F and FIG. 14H showing co-localization atlow magnification. Arrows: Increased TUNEL staining co-localizing withP2X₇ immunoreactivity in epidermal cells of the basal/parabasal layers(horizontal arrow) and of epidermal hair shaft cells (vertical arrow).

FIG. 14K: Overlay of FIG. 14E and FIG. 14G showing co-localization athigh magnification.

FIG. 14L: Overlay of FIG. 14F and FIG. 14H showing co-localization athigh magnification.

FIGS. 15A-15H present exemplary data showing the effects of treatmentswith BzATP on P2X₇ expression and apoptosis in DMBA/TPA-induced skinpapillomas FIGS. (15A-15D) and cancers (FIGS. 15E-15H). Treatment isdescribed herein as Experiment-4. Assays were repeated 4 times withsimilar trends. FIGS. 15C, 15D, 15G, & 15H are parallel cross sectionsto FIGS. 15A, 15B, 15E, & 15F, respectively.

FIG. 16 presents exemplary data showing the effects of BzATP onapoptosis in cultured mouse keratinocytes. In all experiments, levels ofapoptosis were normalized to an arbitrary value of 2 AU in controlcells. AU=arbitrary units.

FIG. 16A: BzATP dose-response effect in cultured mouse normal(wild-type, C57B1) keratinocytes (means±SD, n=3). Cells were treatedwith one of the indicated concentrations of BzATP for 8 hours. Changesin apoptosis were determined in terms of solubilized DNA

FIG. 16B: Cultured mouse normal keratinocytes (wild-type, C57B1) werepre-treated with 100 μM anti-sense P2X₇ oligonucleotides (ASO) orrandom-control P2X₇ oligonucleotides (RCO) for 14 hours followed by 8hours treatment with 100 μM BzATP. Control=cells treated with thevehicle of the ASO. Values are means (±SD) of 3 experiments for eachcondition. Insert: Western immunoblot with anti-P2X₇ antibody of lysatesof cells treated with ASO or RCO (n=2). Changes in apoptosis weredetermined in terms of solubilized DNA

FIG. 16C: BzATP time-response effect in cultured mouse normalkeratinocytes (wild-type, C57B1; filled triangles), or in keratinocytesobtained from C57B1 background P2X₇-deficient mice (P2X₇ ^(−/−)Pfizer;empty circles; or P2X₇ ^(−/−)GSK; filled circles). Values are means(±SD) of 3 experiments for each condition. Changes in apoptosis weredetermined using cell-death detection ELISA.

FIG. 17 presents exemplary data showing the effects of BzATP in mousewild-type normal keratinocytes. BzATP was added (arrows) at 100 μM. Ca²⁺_(o)=extracellular calcium.

FIG. 17A: Time course of changes in cytosolic calcium (ΔCa²⁺ _(i)), inmedium containing 1.2 mM Ca²⁺ (upper trace) or 1.2 mM Ca2+ plus 1.2 mMEGTA (lower trace).

FIG. 17B: Time course of influx of ethidium bromide (Eth-Br).

FIG. 17C: Dose response profile comparison between ΔCa²⁺ _(i) and Eth-Brinflux. Cells were treated with each BzATP concentration for 8 hours.Levels of ΔCa²⁺ _(i) (empty circles) were determined 2 min after addingBzATP; changes in Eth-Br fluorescence (filled circles) were determined 5min after adding BzATP. Values=means±SD. Experiments were repeated 3times. AU=arbitrary units.

FIG. 18 presents exemplary data showing the effects of anti-sense P2X₇oligonucleotides on BzATP treatment of cultured mouse wild-type normalkeratinocytes pretreated with 100 μM anti-sense P2X₇ oligonucleotides(ASO) or random-control P2X₇ oligonucleotides (RCO) for 14 hoursfollowed by 8 hours treatment with 100 μM BzATP. Control=cells treatedwith the vehicle of the ASO. The experiments were repeated twice withsimilar trends.

FIG. 18A: Changes in cytosolic calcium (ΔCa²⁺ _(i)).

FIG. 18B: Influx of ethidium bromide (Eth-Br).

FIG. 19A presents exemplary data showing the dependence of BzATP-inducedapoptosis on extracellular calcium (Ca²⁺ _(o)). Cultured mouse wild-typenormal keratinocytes were shifted for 10 minutes to medium containingone of the indicated Ca²⁺ _(o) concentrations. Control (physiological)level of Ca²⁺ _(o) was 1.2 mM, and levels of Ca²⁺ _(o) were modulated byadding 1.2 mM EGTA. Cells were treated with 100 μM BzATP, or the vehicle(Control), and changes in apoptosis were determined after 8 hours.Apoptosis were determined in terms of solubilized DNA. Values are means(±SD) of 3 experiments. Levels of apoptosis were normalized to anarbitrary value of 2 in non-treated cells. AU=arbitrary units. *=p<0.01as compared to Ca²⁺ _(o) 1.2 mM.

FIG. 19B presents exemplary data showing modulation of BzATP-inducedapoptosis (100 μM, 8 hours) in mouse wild-type normal keratinocytes by50 μM caspase inhibitors incubated for 8 hours. Apoptosis weredetermined in terms of solubilized DNA. Values are means (±SD) of 3experiments. Levels of apoptosis were normalized to an arbitrary valueof 2 in non-treated cells. AU=arbitrary units. *=p<0.01 compared tocontrol.

FIG. 20 presents exemplary data showing the effects of pre-treatmentwith anti-sense P2X₇ oligonucleotides (ASO) or random-control P2X₇oligonucleotides (RCO) (both at 100 μM for 14 hours), and of treatmentswith BzATP (100 μM, 8 hours) on [³H]-thymidine incorporation in mousewild-type normal keratinocytes (values are means±SD, n=4).

FIG. 21A shows one embodiment of a predicted structure of a P2X₇receptor monomer, showing a short intracellular N-terminus; twotransmembrane (TM) segments containing the extracellular domain; and thelong intracellular C-terminus.

FIG. 21B and FIG. 21C show structural embodiments of P2X₇ receptorligands ATP and BzATP.

FIG. 21D illustrated one embodiment of the expression, mechanism ofaction, and regulation of the P2X₇ receptor. The functional P2X₇receptor is a glycosylated G-coupled membrane-bound protein and itsnatural ligand is ATP. Activation stimulates GRK-3-mediatedphosphorylation of the receptor (pi-P2X₇-R), and recruitment ofβ-arrestin-2 (β-arr-2) to the plasma membrane. The activated receptorcan induce stimulation of one or more signaling pathways. β-arrestinbinding facilitates uncoupling of the receptor from the heterotrimeric Gproteins (a,b,g), and targets the receptor in a dynamin-relatedmechanism to clathrin-coated pits for endocytosis. Endocytosis can befollowed by receptor sequestration into various cellular domains,recycling, and degradation. Glycosylation of the P2X₇ receptor iscontrolled by β2-adrenoceptor (β-AR)-activation of PKA, resulting inde-glycosylation of the P2X₇ receptor and enhanced receptor degradation.The PKA effect is regulated by the action of the EGF-EGFR system; itinvolves facilitated, PI3K-dependent inhibition of β-AR internalization,and facilitated β-AR recycling, thereby increasing the pool of β-ARs inthe plasma membrane that are available for activation upon ligandbinding.

FIG. 21E illustrates one embodiment of ligand-induced pore formationinvolving homo(tri)-oligomerization of P2X₇ monomers. Cells express thefull-length receptor (square), as well as truncated forms of the P2X₇,e.g. the P2X_(7-j) variant (triangle) that can hetero-oligomerize withthe full-length P2X₇ form and produce non-functional pores. Data in hostcells co-expressing the P2X₇ plus the P2X_(7-j) suggested the formation,shown in the figure, of four types of complexes (numbers in parenthesesare the order of expression). F. Pathway of physiological P2X₇-mediatedapoptosis in epithelial cells. Activation of the P2X₇ receptor (P2X₇R)by ATP induces pore formation and uncontrolled influx of Ca²⁺,triggering permeabilization of the mitochondria and activation ofcaspase 9/7/3-mediated apoptosis.

FIG. 22 presents a schema of possible mechanisms that regulate andcontrol the expression, activation, and dynamics of the P2X₇ receptor.

FIG. 23A: Design of the study, Fu et al., “Activation of P2X(7)-mediatedapoptosis Inhibits DMBA/TPA-induced formation of skin papillomas andcancer in mice” BMC Cancer 9:114 (2009). Drugs were applied locally onthe shaved dorsal skin of mice at the indicated times. Shown are thephases of papilloma and skin cancers formation in response to thetreatments with DMBA and TPA.

FIGS. 23B-E: Representative pictures of DMBA/TPA-induced skin lesionsin-vivo.

FIGS. 23F-I: Representative skin cross-sections, evaluatedhistologically by H&E, of normal and DMBA/TPA-induced lesions in-vivo.

FIG. 24 present a summary of the effects of local treatments withDMBA/TPA (continuous lines) or DMBA/TPA+BzATP (broken lines) on thetime-to-event of cancer state (A) and on the animals' survival rates(B).

FIG. 25 presents illustrative data showing P2X₇ immunoreactivity inmouse normal skin (A), papilloma (C), and skin cancer tissues (E); B,D,Fare parallel cross sections, respectively, stained by H&E. G. Analysisof P2X7 immunoreactivity compared among paired histologically normal andcancerous tissues. Bars are means (±SD, n=5). H. P2X7 mRNA levels(relative to GAPDH mRNA) (means±SD, n=3) in histologically normal andcancerous tissues. In G and H data were normalized to an arbitrary valueof 10. *p<0.01. AU—arbitrary units.

FIG. 26 presents illustrative data showing effects of local treatmentwith BzATP in-vivo on skin apoptosis. A-J: Mice were treated for 16weeks either with BzATP, applied locally twice a week on the shaveddorsal skin (F-J), or with the vehicle (Control, A-E). At the end of theexperiment animals were euthanized and strips were obtained from eachanimal dorsal skin areas for H&E (A,F); and for P2X7 immunostaining plusTUNEL (apoptosis) co-staining (B-E, G-J). Arrows in I show increasedTUNEL staining co-localizing with P2X7 immunoreactivity in epidermalcells of the basal/parabasal layers (horizontal arrow) and of epidermalhair shaft cells (vertical arrow). E and J are higher magnification of Dand I. K-N: Mice were treated with BzATP, applied locally twice a weekfor 4 weeks on the shaved anterior skin area, and with the vehicle(Control) applied in parallel on the shaved posterior skin area. At theend of the experiment animals were euthanized and skin cross sectionswere generated from each animal for H&E (K,M) and TUNEL (L,N) staining.Arrows in N show enhanced TUNEL staining in epidermal cells of thebasal/parabasal layers (horizontal arrow), and of epidermal hair shaftcells (vertical arrow). O-V: Effects of treatments with BzATP on P2X7expression and apoptosis in DMBA/TPA-induced skin papillomas (O-R) andcancers (S-V). P,R,T,V are parallel cross sections to O,Q,S,U,respectively.

FIG. 27 demonstrates one embodiment of a proposed schema of a CpG-rich547 nt DNA region (+26/+573) downstream of an active promoter of a P2X₇gene. Filled ellipses denote CpG sites that were found experimentally toinhibit P2X₇ transcription. Upwards pointing arrows denote CpG sitesthat were found experimentally to be hypermethylated in culturedcervical cells and in cervix epithelial tissues in vivo. Hatched squaresare cis regions that were found experimentally to possess transcriptionenhancer activity. Horizontal bi-directional arrows show four putativesites within the cis-enhancer regions that were found to formDNA-protein complexes.

FIG. 28 presents one embodiment of a nucleotide sequence of a 5′ regionof human P2X₇ (SEQ TD NO: 62), containing an active promoter (whitesymbols on black background, nt −158/+32); a 547 nt CpG-rich region(underlined, nt-+26/+573) downstream of the promoter; Exon 1 (underlinedand italics, nt +92/+216); and the proximal part of intron-1 (distal toExon 1, underlined, beginning at nt +217). Nucleotides were numberedrelative to the Transcription Initiation Start Site (TpIS) (+1; nt 1683according to GenBank Y12851). TpISs and TATA-like sequences within theactive promoter bases/regions are bolded and doubly underlined. CpGdinucleotides are bolded and doubly underlined. Vertical thick emptyarrows point to MaeII-sensitive CpG sites (nt +193/+194, +211/+212, and+330/+331). The vertical thick filled arrow points to a BstUI-sensitiveCpG-CpG site (nt +461/+462 and +463/+464). For DNA methylationexperiments the 547 nt CpG-rich region was subdivided into Segment-1 (nt+26/+247), Segment-2 (nt +223/+399), and Segment-3 (nt +352/+573).

FIG. 29 presents one embodiment of an elucidation of a P2X₇ activepromoter region.

FIG. 29A: cDNA fragments corresponding to regions within a 1.7 kb DNAsegment of the 5′ region of the human P2X₇ gene were inserted intoluciferase vector; the P2X₇-luciferase reporters were transfected intoHEK293 and P2X₇ promoter activity was determined in terms of changes inluciferase activity (Fluc/Rluc). Data (means±SD, 3-5 experiments intriplicates) were normalized (=1) to Fluc/Rluc recorded in cellstransfected with empty vector. *-p<0.01 compared to the rest.

FIG. 29B: Confirmation of P2X₇ TpIS. Two potential TpISs and theirrelated TATA-like regions were mutated and effects on P2X₇ transcriptionwere determined as in FIG. 29A (means±SD, 3-5 experiments intriplicates). *-p<0.01 as compared to −158/+32.

FIG. 29C: P2X₇ transcription is modulated by effectors downstream of theactive promoter. P2X₇ −158/+32 or −158/+573 luciferase reporters weretransfected into HEK293 cells and P2X₇ promoter activity was determinedin terms of changes in luciferase activity (Fluc/Rluc, upper panel) orin terms of changes in Fluc/GAPDH mRNA (lower panel). Shown are means(±SD) of 1-3 experiments in triplicates. Data of Fluc/GAPDH mRNA werenormalized (=0) to those recorded in cells transfected with emptyvector. *-p<0.01.

FIG. 30 presents exemplary data showing effects of treatments withAza-dC (1 μM) on P2X₇ mRNA expression in hEVEC and HeLa cells. Data in Aand B were normalized (=1) to levels in hEVEC cells at t=0. Means±SD of3-6 experiments in triplicates.

FIG. 30A: Steady-state levels of P2X₇ mRNA. (p<0.01)

FIG. 30B: P2X₇ receptor protein levels. Insert: Immunofluorescence data@ 20×. (p<0.01)

FIG. 30C: BzATP-induced apoptosis (in arbitrary units [A.U.]) followingAza-dC. The degree of apoptosis (in arbitrary units [A.U.]) wasnormalized to levels determined in non-treated cells. *-p<0.05;**-p<0.01.

FIG. 31 presents exemplary data showing effects of hypermethylation andde-methylation on changes in transcription in HEK293 cells transfectedwith the luciferase −158/+32 or −158/+573 reporter constructs (means±SDof two experiments in triplicates).

Hypermethylation assays were done by incubating the test plasmids priorto transfections with the CpG-Methylase M.SssI and changes intranscription were determined in terms of changes in luciferase activity(Fluc/Rluc). De-methylation assays were done by treating transfectedcells with Aza-dC (1 μM for 48 hours). Changes in transcription weredetermined in terms of changes in Fluc/GAPDH mRNA levels. Data werenormalized (=1) to levels in control cells. *-p<0.01.

FIG. 32 presents several embodiments of constructs utilized to elucidatetranscription regulatory cis-elements within a CpG-rich 547 nt regiondownstream of the P2X₇ promoter. Constructs comprise a P2X₇ activepromoter attached with one of the shown segments and were inserted intoa luciferase vector and transfected into HEK293 cells. Promoter activitywas determined in terms of changes in luciferase activity (Fluc/Rluc,means±SD, of two experiments in triplicates).

FIG. 32A: cDNA fragments were constructed containing the P2X₇ activepromoter (nt −158/+32) attached with one of the shown segments of the547 nt region downstream of the promoter. *-p<0.01 compared to −158/+32;**-p<0.05-0.01 compared to −158/+232.

FIG. 32B: Effects of mutations in the CpG sites within the 547 nt regiondownstream of the promoter on P2X₇ transcription. WT—wild-type. *-p<0.01compared to the wild-type sequence in each case.

FIG. 33A presents exemplary data showing effects of treatment withAza-dC in HeLa cells (1 μM, 48 hours) on methylation of cytosines in CpGsites +193/+194 (and/or +211/+212), +330/+331, and +461/+462 (and/or+463+464), within Segments 1, 2, and 3 respectively of a 547 nt regiondownstream of the P2X7 active promoter. Methylation of cytosines in CpGsites was determined in terms of cleavage at CpG sites using the genomicDNA bisulfite conversion method followed by gene specific PCR andrestriction enzyme cutting. Left-pointing arrows show bandscorresponding to uncleaved (broken lines) and cleaved fractions(continuous lines) at the CpG sites. M—markers. Controls were aliquotsof human placental genomic DNA (H.G.—DNA) treated in vitro with the CpGmethylase SssI.

FIG. 33B presents exemplary data from aliquots of human placentalgenomic DNA were mixed with different molar concentrations of SssI andthe degree of cleavage at CpG sites +193/+194 (and/or +211/+212) withinSegment-1. Data were normalized to the effect (100%) obtained in areaction mixture containing 1 μg DNA.

FIGS. 33C-E present exemplary data showing methylation status ofcytosines in CpG sites in cultured human epithelial uterine cervicalcells.

FIGS. 33F-H present exemplary data showing methylation status ofcytosines in CpG sites in human uterine cervix tissues in-vivo.

FIG. 33I presents exemplary data showing degree of restriction enzymecleavage at CpG sites in tissues of human cervix. Data were compiledfrom 10 sets of paired cervical specimens, including in each case normaland squamous cell carcinoma tissues. Available for analysis were 9 casesfor Segments 1 and 3, and 8 cases for Segment 2. Lines connect pairedtissues (Normal [N] and Cancer [Ca]) from the same patient.

FIG. 34 presents exemplary data showing an elucidation of representativeDNA-protein binding within the 547 nt region downstream of the P2X₇promoter. The indicated cDNA fragments are depicted herein. See, Table4. Electrophoretic mobility shift assays (EMSA) were used to detectDNA-protein complexes. Right-pointing arrows indicate shifted bands. Theexperiment was repeated twice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the field of treatment andprevention of cancer. Cancer is believed to be a disease ofoverproliferation, wherein the present invention provides a method toreduce this overproliferation. For example, the methods herein result inthe induction of intracellular apoptosis, thereby resulting in theprevention of cancerous growth and/or a reduction in cancer tumor sizeand number. The administration of a specific membrane receptor agonist(i.e., for example, a P2X₇ receptor agonist) is shown herein to reducecancer growth and progression.

Anti-apoptotic mechanisms may contribute to the development of cancer.The P2X₇ system is believed to be a pro-apoptosis modulator inepithelial cells, and augmentation of P2X₇-mediated apoptosis has beenproposed as a pharmacological modality for chemoprevention and treatmentof epithelial cancers.

The growing understanding of mechanisms of P2X₇-mediated apoptosis hasgenerated a strategy for targeting directly and specifically skinneoplasia. Although it is not necessary to understand the mechanism ofan invention, it is believed that the data presented herein linkdirectly, for the first time, an up-regulation of apoptosis with cancerprevention and treatment. For example, significant antitumor efficacyhas been achieved in a rodent cancer model, and it is likely thatcompounds affecting P2X₇-control of apoptosis are useful for preventingand treating cancer (i.e., for example, epithelial cancer).

The data discussed herein demonstrates that a pharmacological activationof P2X₇-mediated apoptosis (i.e., for example, by a P2X₇-receptoragonist such as BzATP) inhibits chemically-induced (i.e., for example,by DMBA/TPA) formation of epithelial papillomas and/or squamousspindle-cell carcinomas. P2X₇ agonists, such as BzATP, are believed toincrease intracellular apoptosis.

In one embodiment, the present invention contemplates a compositioncomprising at least one compound capable of activating P2X₇-inducedapoptosis. In one embodiment, the compound comprises BzATP. Although itis not necessary to understand the mechanism of an invention, it isbelieved that, in vivo, P2X₇-induced apoptosis may control thedevelopment and progression of neoplasias. In one embodiment, theneoplasias comprise epithelial neoplasias. In one embodiment, theepithelial neoplasia is derived from an ectoderm layer. In oneembodiment, the ectoderm layer neoplasia is selected from the groupconsisting of a skin neoplasia and a breast neoplasia. In oneembodiment, the epithelial neoplasia is derived from a uro-genitalsinus. In one embodiment, the uro-genital sinus neoplasia comprises abladder neoplasia. In one embodiment, the epithelial neoplasia isderived from a distal paramesonephric duct. In one embodiment, thedistal paramesonephric duct neoplasia is selected from the groupconsisting of a uterine cervix neoplasia and an endometrium neoplasia.Li et al., “The P2X₇ Receptor: A novel biomarker of uterine epithelialcancers” Cancer Epidemiol Biomarkers Preven 2006, 15:1-8; Li et al.,“Decreased expression of P2X7 in endometrial epithelial pre-cancerousand cancer cells” Gynecol Oncology 2007, 106:233-243; Zhou et al.,“Micro-RNAs miR-186 and miR-150 downregulate expression of thepro-apoptotic purinergic P2X₇ receptor by activation of instabilitysites at the 3′-untranslated region of the gene that decreasesteady-state levels of the transcript” J Biol Chem 2008,283:28274-28286; and Li et al., “P2X₇ receptor expression is decreasedin epithelial cancer cells of ectodermal, uro-genital sinus, and distalparamesonephricduct origin” (Submitted, 2009).

I. Cancer

Cancer is believed to be an uncontrolled growth of abnormal cells in thebody. Cancerous cells are also called malignant cells and are derivedfrom normal cells in the body. Cancer appears to occur when the growthof cells in the body is out of control and cells divide too quickly. Itcan also occur when cells “forget” how to die (i.e., for example,reduced apoptosis). There are many different kinds of cancers. Cancercan develop in almost any organ or tissue, including, but not limitedto, the lung, colon, breast, skin, bones, or nerve tissue.

There are many causes of cancers, including, but not limited to, benzeneand other chemicals, poisonous mushrooms and a type of poison that cangrow on peanut plants (i.e., for example, aflatoxins), viruses,radiation, sunlight, or tobacco. However, the cause of many cancersremains unknown. The most common cancers in men in the United Statesinclude, but are not limited to skin cancer, prostate cancer, lungcancer, and colon cancer. In women in the U.S., the most common cancersinclude, but are not limited to, breast cancer, skin cancer, lungcancer, and colon cancer.

Some other types of cancers include, but are not limited to, braincancer, cervical cancer, Hodgkin's lymphoma, kidney cancer, leukemia,liver cancer, Non-Hodgkin's lymphoma, ovarian cancer, skin cancer,testicular cancer, thyroid cancer, or uterine cancer. Symptoms of cancerdepend on the type and location of the tumor. For example, lung cancercan cause coughing, shortness of breath, or chest pain. Colon canceroften causes diarrhea, constipation, and blood in the stool. Somecancers may not have any symptoms at all. In certain cancers, such asgallbladder cancer, symptoms often do not start until the disease hasreached an advanced stage. The following symptoms can occur with mostcancers: chills, fatigue, fever, loss of appetite, malaise, nightsweats, or weight loss.

Common tests to identify cancer may include, but are not limited to,biopsy, blood chemistries x-ray, complete blood count, computerizedtomography scan, or magnetic resonance imaging scan.

Conventional treatment varies based on the type of cancer and its stage.The stage of a cancer refers to how much it has grown and whether thetumor has spread from its original location. If the cancer is confinedto one location and has not spread, current treatments are orientedtowards surgery, radiation and/or chemotherapy. This is often the casewith skin cancers, as well as cancers of the lung, breast, and colon.

A. Epithelial Cancers

Epithelial cancers are common and usually display aggressive and fatalbiological-clinical behavior. Epithelia are tissues that line bodysurfaces. Although it is not necessary to understand the mechanism of aninvention, it is believed that the present invention will lead to betterunderstanding of how epithelial cancers develop. In one embodiment, thepresent invention contemplates a method for detecting cancers at earlystage of development, consequently resulting in earlier treatment andimproved survival rates. In one embodiment, the present inventioncontemplates methods of treating epithelial cancers. In one embodiment,the present invention contemplates methods of preventing epithelialcancers (i.e., for example, prophylactic treatments).

Epithelial cancers are thought to be common and can display aggressiveand potentially fatal biological clinical behavior. Although it is notnecessary to understand the mechanism of an invention, it is believedthat some embodiments of the present invention could lead to: i)improved understanding of epithelial cancer development; ii) improvedearly cancer detection; iii) improved early cancer treatment; iv) newmodalities and directions for cancer treatments; and v) improvedepithelial cancer prevention.

Cancer development is believed associated with inactivation oftumor-controlling genes, including tumor suppressor andapoptosis-related genes. Inactivation of genes can be the result ofallelic loss or loss-of-heterozygosity chromosomal sites due to genemutations, deletions, and genomic rearrangements. Some cancers exhibit anumber of genomic alterations including monoallelic hemizygous deletionsat 4p15.3, 10q24, 5q35, 3p12.3, and 11q24. Wistuba et al., “Deletions ofchromosome 3p are frequent and early events in the pathogenesis ofuterine cervical carcinoma” Cancer Res. 57:3154-3158 (1997); Chu et al.,“Monoclonality and surface lesion specific microsatellite alterations inpremalignant and malignant neoplasia of uterine cervix: a local fieldeffect of genomic instability and clonal evolution” Genes ChromosomesCancer 24:127-134 (1999); and Hamoudi et al., “Identification of novelprognostic markers in cervical intraepithelial neoplasia using lDMAS(loh data management and analysis software)” BMC Bioinformatics 6:18(2005). Alternatively, other studies demonstrate a possible loss oftumor suppressor gene on chromosome 11q23. Lai et al., “Hypermethylationof two consecutive tumor suppressor genes, BLU and RASSF1A, located at3p21.3 in cervical neoplasias” Gynecol Oncol. 104:629-635 (2007).

II. Apoptosis

The current theory of epithelial cell growth predicts regulation by theconcerted actions of mitogenic stimuli and apoptosis [1,2]. Apoptosis isa homeostatic process orchestrated by the host's genome of selectivecell deletion without stimulating inflammatory response [3-5]. Earlierstudies showed that apoptosis is activated in response to noxiousstimuli e.g. starvation, inflammation, infection, irradiation, etc. Morerecent data suggested a physiological role for apoptosis, including thecontrol of tissue development and differentiation, regulation ofmitogenic effects, and control of cell death and loss of tissue withaging, and dysregulation of apoptotic cell-death has been implicated instates of disease [6].

Apoptosis is believed to be a process of programmed cell death that mayoccur in multicellular organisms. Programmed cell death involves aseries of biochemical events leading to a characteristic cell morphologyand death, in more specific terms, a series of biochemical events thatlead to a variety of morphological changes, including blebbing, changesto the cell membrane such as loss of membrane asymmetry and attachment,cell shrinkage, nuclear fragmentation, chromatin condensation, andchromosomal DNA fragmentation. Processes of disposal of cellular debriswhose results do not damage the organism differentiate apoptosis fromnecrosis.

In contrast to necrosis, which is a form of traumatic cell death thatresults from acute cellular injury, apoptosis, in general, confersadvantages during an organism's life cycle. For example, thedifferentiation of fingers and toes in a developing human embryo occursbecause cells between the fingers apoptose; the result is that thedigits are separate. Between 50 billion and 70 billion cells die eachday due to apoptosis in the average human adult. For an average childbetween the ages of 8 and 14, approximately 20 billion to 30 billioncells die a day. In a year, this amounts to the proliferation andsubsequent destruction of a mass of cells equal to an individual's bodyweight. Excessive apoptosis causes hypotrophy, such as in ischemicdamage, whereas an insufficient amount results in uncontrolled cellproliferation, such as cancer.

Apoptosis may occur when a cell is damaged beyond repair, infected witha virus, or undergoing stressful conditions such as starvation. Damageto DNA from ionizing radiation or toxic chemicals can also induceapoptosis via the actions of the tumour-suppressing gene p53. The“decision” for apoptosis can come from the cell itself, from thesurrounding tissue, or from a cell that is part of the immune system. Inthese cases, apoptosis functions to remove the damaged cell, preventingit from sapping further nutrients from the organism, or halting furtherspread of viral infection.

As discussed further below, apoptosis may also play a role in preventingcancer. If a cell is unable to undergo apoptosis because of mutation orbiochemical inhibition, it continues to divide and may develop into atumor. For example, infection by papillomaviruses causes a viral gene tointerfere with the cell's p53 protein, an important member of theapoptotic pathway. This interference in the apoptotic capability of thecell plays a role in the development of cervical cancer.

In an adult organism, the number of cells is kept relatively constantthrough cell death and division (i.e., proliferation). Cells must bereplaced when they malfunction or become diseased, but proliferationmust be offset by cell death. This control mechanism is part of thehomeostasis required by living organisms to maintain their internalstates within certain limits. Homeostasis is achieved when the rate ofmitosis (cell division) in the tissue is balanced by cell death. If thisequilibrium is disturbed, one of two potentially fatal disorders mayoccur: i) the cells are dividing faster than they die, effectivelydeveloping a tumor; or ii) the cells are dividing slower than they die,causing cell loss.

Homeostasis involves a complex series of reactions, an ongoing processinside an organism that calls for different types of cell signaling. Anyimpairment can cause a disease. For example, dysregulation of signalingpathway has been implicated in several forms of cancer. The pathway,which conveys an anti-apoptotic signal, has been found to be activatedin pancreatic adenocarcinoma tissues.

A. Mechanisms of Apoptosis

Histologically, apoptosis may be characterized by DNA fragmentation,chromatin condensation, membrane bleeding, cell detachment from theextracellular matrix, cell rounding and shrinking, and alterations inplasma membrane lipid organization. Usually, the final stages ofapoptosis are induced by a series of proteolytic enzymes termedcaspases, which cleave and activate each other in a cascade ofproteolysis [14], terminating with the effector caspases 7 and 3 [15].

Several cellular pathways are involved in the activation of the caspasefamily of proteases and the induction of apoptosis. In one embodiment,the present invention contemplates a method wherein apoptosis mayinvolve pathways including, but not limited to: a) the intrinsicmitochondrial pathway; or b) the extrinsic death-receptor pathway. [16]

Apoptosis via the intrinsic pathway is characterized predominantly bymitochondrial changes. Effects are triggered by stimuli that causemitochondrial disturbances and DNA damage (such as cancer therapeuticagents and ionizing irradiation), oxidative stress, hypoxia, celldetachment, and cellular distress [17]. Signals from these diversestimuli converge upon the mitochondria, where propagation of theapoptotic signal is regulated by proteins that either promote (e.g. Bax,Bak, Bok, Bad, Bid, Bik, Bim, Bcl-Xs, Krk, Mtd, Nip3, Nix, Noxa, andBcl-B) or suppress apoptosis (e.g. Bcl-2, Bcl-XL, Mcl-1, Bfl-1/Al,Bcl-W, and Bcl-G) [18,19]. Pro-apoptotic signals triggerpermeabilization of the mitochondrial outer membrane, and facilitate therelease of proteins from the mitochondrial intermembranous space intothe cytoplasm, including cytochrome c and Smac/Diablo. The releasedcytochrome c then binds the caspase adaptor apoptoticprotease-activating factor-1 (Apaf-1), thereby activating procaspase 9and forming the apoptosome complex [20]. The apoptosome activatesseveral downstream effector caspases, such as caspases 6, 7 and 3,leading to DNA fragmentation and cell death [21,22]. The effects ofpro-apoptotic signals can be modulated by inhibitors of apoptosisproteins (IAPs), e.g. c-IAP1, c-IAP2, NAIP, Survivin, XIAP, Bruce,ILP-2, and Livin [23]. IAPs directly inhibit caspases and/or catalyzetheir ubiquitination and proteaseome-mediated degradation. This balanceis finely regulated by endogenous inhibitors of IAPs, such as SMAC andHtrA2, which compete with active caspases to bind to IAP [24].Anti-apoptotic signals such as Bcl-XL can bind and inactivate Apaf-1,and stimulate the release of Smac/DIABLO proteins from the mitochondria,thereby inactivating the IAPs [25].

The extrinsic pathway of apoptosis is a mechanism by which cells of theimmune system trigger apoptosis in ‘unhealthy’ cells throughligand-mediated activation of cell surface death-mediating receptors,such as TNF Receptor 1 (TNFR1), TNF Receptor 2 (TNFR2), CD95/Fas/Apo1,and Death Receptors (tumor necrosis factor-related apoptosis-inducingligand [TRAIL]-TRAIL receptors) 3-6 (DR3-6) [15,17].

Binding of these receptors by their respective ligands leads to receptoroligomerization and recruitment of death signal adaptor proteins. Forexample, binding of Fas ligand (Fas-L) to Fas, or TRAIL to TRAIL-R1 [26]leads to recruitment of FADD (Fas-associated death domain), and bindingof TNF to TNFR1 leads to recruitment of TRADD (TNFR-associated deathdomain) [22]. The oligomerized receptors and recruited FADD or TRADDform a complex termed DISC (death-inducing signaling complex), which canbind to initiator caspases (caspase 8 and 10), followed by triggeringthe activation of caspases 7 and 3, and leading to apoptosis [13,15].

Recent studies underscore deficiencies in the arbitrary classificationof intrinsic and extrinsic apoptosis pathways. First, some signals canactivate both pathways, and an extensive crosstalk exists between thesetwo apoptosis pathways. For instance, the transcription factor NF-kβ canactivate the transcription of anti-apoptotic genes such as FLIP, Bcl-XL,XIAP and cIAP1; however, NF-kβ can also enhance the expression ofapoptosis-inducing genes such as Fas, Fas-L, TRAIL-R1 and TRAIL-R2 [27].

Recent data has further suggested that the extrinsic death-receptorpathway is not limited to cells of the immune system, and that growthcontrol of ‘unhealthy’ cells operates in most/all tissues containingproliferating cells. Thus, the P2X₇ receptor mechanism controls growthof certain types of epithelial cells, under normal physiologicalconditions, and, as contemplated herein, impaired P2X₇-mediatedapoptosis could contribute to the neoplastic transformation in thosetissues. Those discoveries suggest a physiological role for apoptosis inmaintaining cellular homeostasis.

The improved understanding of apoptosis has provided a basis fortargeted therapies that can induce death of cancer cells or sensitizethem to established cytotoxic agents and radiation therapy [24,28,29].Previous reports outlined agents and methods that suggest selectiveinduction of apoptosis in cancer cells might be potentially useful incancer therapy [reviewed in 13,24,25,30-32]. Such apopotic mechanismsinclude, but are not limited to, i) activation of the cell surface deathreceptors Fas, TRAIL and TNF receptors; ii) inhibition of cell survivalsignaling via EGFR, MAPK and PI3K; iii) altering the balance betweenpro-apoptotic and anti-apoptotic members of the Bcl-2 family; iv)down-regulating anti-apoptosis proteins such as XIAP, surviving andc-IAP2; e) proteasome inhibitors; f) nonsteroidal anti-inflammatorydrugs (NSAIDs) and COX-2 inhibitors; g) peroxisomeproliferator-activated receptor (PPAR) ligands; or h) DNA methylation.

Despite the expanse of present research, however, only a small number oftherapies directly targeting the apoptotic pathways have advanced intoclinical testing, and none have yet achieved approval by the UnitedStates Food And Drug Administration. Of the clinical trials that wereinitiated using agents such as those listed above, many were of limitedvalue because of problems including, but not limited to: i) low efficacy[33]; ii) toxicity [34]; iii) presence of decoy receptors (DcR1, DcR2,and osteoprotegerin) which bind TRAIL and inhibit apoptosis [35]; iv)concerns of inducing immunodeficiency with hypogammaglobulinemia; or v)predisposition to develop lymphomas [36].

B. Apoptosis and Cancer

Defective apoptosis may play a role in the development of cancers [7-9].In fact, one of the hallmarks of cancer is the development of mechanismsthat evade apoptosis, and the loss of pro-apoptotic signals and gain ofanti-apoptotic mechanisms contribute to tumorigenesis and the cancerphenotype. Thus, defective apoptotic mechanisms allow geneticallyunstable cancer cells to avoid elimination and confer resistance tocancer treatments [10-11]. Since apoptosis does not elicit inflammatoryor immune response, this type of cell death is the preferred way ofcancer cell killing by various treatments. The selective induction ofapoptosis in cancer cells is emerging as a promising therapeuticapproach for many cancers [13], and modulating the apoptotic pathwaysmay be involved in mechanisms including, but not limited to, i) inducingtumor-cell death; ii) increasing responses to chemotherapy, radiotherapyand other targeted therapies; or iii) prevention of the neoplastictransformation.

Levels of the functional P2X₇ receptor in cancer epithelial cells of theectoderm, the uro-genital sinus, and the distal paramesonephric duct arereported to be lower compared to normal cells (infra). The lesserexpression of the P2X₇-receptor could be the result of the neoplastictransformation. Thus, in endometrial and bladder cells low expression ofthe P2X₇ receptor was found already in pre-cancerous and early cancerouscells, but not in hyperplastic benign cells [39,82,104]. As the datapresented herein demonstrates, the carcinogenic process could haveinduced reduced expression of the P2X₇ at early stages of cancerdevelopment. Alternatively, the neoplastic transformation could havebeen triggered preferentially in cells expressing low levels of thereceptor. This possibility is supported by data in uterine cervicalepithelia, where low expression of the P2X₇ receptor was found alreadyin dysplastic (precancerous) cells [39,82,104]. Few cases of dysplasiaprogress to cancer [109], so it is possible that low expression of thereceptor preceded the neoplastic transformation. Accordingly, cellsharboring defective P2X₇ expression mechanism have escaped apoptosis,and were rendered susceptible to carcinogenic stimuli and the neoplastictransformation.

In both scenarios, low expression of the P2X₇ receptor could promotecancer development, because decreased apoptosis due to reduced receptorexpression can facilitate the growth of neoplastic cells. A recent studytested the hypothesis that in tissues at risk for undergoing malignanttransformation augmentation of P2X₇-mediated apoptosis could inhibitcancer development [55].

II. P2X Receptor Family

The human P2X₇ receptor gene is localized to chromosome 12q24 andcomprises 13 exons. Buell et al., “Gene structure and chromosomallocalization of the human P2X₇ receptor” Receptors Channels 5:347-354(1998). Some genetic mutations in the P2X₇ receptor gene have beendescribed, but none regarding cervical cancer. Feng et al., “A truncatedP2X₇ receptor variant (P2X_(7-j)) endogenously expressed in cervicalcancer cells antagonizes the full-length P2X₇ receptor throughhetero-oligomerization” J Biol Chem. 281:17228-17237 (2006). Since theoverall prevalence of known chromosomal abnormalities in cervicalcancers is low, genetic mutations cannot be considered the mainetiological factor of the disease.

It has been reported that the P2X₇ receptor may belong to the P2Xsub-family of P2 nucleotide receptors which are membrane-bound,ligand-operated channels. Buell et al, 1996; Soto et al, 1997; Dubyakand el-Moatassim, 1993; Ralevic and Burnstock, 1998; Khakh et al, 2001.For example the nucleotide, adenosine triphosphate (ATP), is believed tobe a naturally occurring P2X₇ receptor ligand. Dubyak and el-Moatassim,1993; Ralevic and Burnstock, 1998. ATP has been reported to beconstitutively secreted by cells wherein ATP levels in extracellularfluids may be present in a low micromolar range. Sperlágh et al, 1998;Grahames et al, 1999; Henriksen and Novak, 2003; Loomis et al, 2003;Wang et al, 2004a. Early studies suggested that, in contrast to othertypes of ATP receptors, activation of the P2X₇ receptor might require arelatively high concentration of ligand. Ralevic et al., “Receptors forpurines and pyrimidines” Pharmacol Rev 1998, 50:413-492. However, thedata shown herein demonstrate that a threshold effect of P2X₇-mediatedapoptosis occurs at nanomolar concentrations of ATP, suggesting that ATPlevels which are present in the extracellular fluid are sufficient toactivate the P2X₇ receptor.

One cellular effect of P2X₇ receptor activation may involve theformation of pores in the plasma membrane. Wang et al., “Anti-apoptoticeffects of estrogen in normal and in cancer human cervical epithelialcells” Endocrinology 2004, 145:5568-5579. For example, in uterineepithelial cells, formation of P2X₇ receptor pores induces apoptosis bya mechanism believed to involve influx of Ca²⁺ via the P2X₇-pores inparallel with an activation of the mitochondrial-caspase-9 pathway.North R A, “Molecular physiology of P2X receptors” Physiol Rev 2002,82:1013-1067; Wang et al., “Anti-apoptotic effects of estrogen in normaland in cancer human cervical epithelial cells” Endocrinology 2004,145:5568-5579; and Feng et al., “A truncated P2X₇ receptor variant(P2X_(7-j)) endogenously expressed in cervical cancer cells antagonizesthe full-length P2X₇ receptor through hetero-oligomerization” J BiolChem 2006, 281:17228-17237. P2X₇ receptor activation by a brief exposureto extracellular ATP has been reported to open cation channels thatapparently allow Ca²⁺, Na⁺ and K⁺ influx. Surprenant et al, 1996.Further, a longer exposure to ATP may induce pore formation in theplasma membrane. Virginio et al, 1999.

The P2X₇ receptor is believed to play a role in cell growth because thereceptor is expressed by proliferating cells. Li et al., “The P2X₇Receptor: A novel biomarker of uterine epithelial cancers” CancerEpidemiol Biomarkers Preven 2006, 15:1-8. Further, it has been reportedthat activation of the P2X₇ receptor induces apoptosis thereby having aregulatory impact on cell growth. Wang et al., “EGF facilitatesepinephrine inhibition of P2X₇-receptor mediated pore formation andapoptosis: a novel signaling network” Endocrinology 2005, 146:164-174.

P2X₇-mediated apoptosis may also involve intracellular signalingmechanisms including, but not limited to, IL-1β (Ferrari et al, 1997a),TRAIL (Aggarwal et al, 1999), p38/JNK/SAPK (Humphreys et al, 2000) andNF-κB (Ferrari et al, 1997b). Other cellular effects of P2X₇ receptoractivation may be determined by receptor synthesis (Guerra et al, 2003),glycosylation (Feng et al, 2005), trafficking and plasma membranelocalization (Li et al, 2000; Bobanovic et al, 2002; Gu et al, 2000),oligomerization (Khakh et al, 2001; Feng et al, 2006), andpost-activation internalization, recycling and degradation (Wang et al2005; Feng et al, 2006).

Until recently, relatively little was known about the in vivo biologicalrole of the P2X₇ receptor. Earlier studies suggested involvement of theP2X₇ receptor in inflammatory and immune processes since the receptor isexpressed in the islets of Langerhans and inflammatory dendriticepidermal cells and in cultured immature dendritic epidermal cells.Georgiou et al. 2005; Mutini et al, 1999. Overexpression of P2X₇ wasfound in lesional skin of psoriasis and atopic dermatitis, where anintense P2X₇ immunoreactivity was confined to the cell membrane of thebasal layer. Pastore et al, 2007. P2X₇ has been suggested to play a rolein chemokine secretion by normal keratinocytes but available data areinconsistent. For example, one study reported that the treatment ofcultured normal keratinocytes with the P2X₇ specific agonist2′,3′-0-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP) increasedIL-6 release, while a second report found that BzATP decreased chemokinesecretion. Inoue et al, 2007; and Pastore et al, 2007, respectively.

Studies have also suggested a role for P2X₇ in the control of epidermalgrowth, but most studies were observational. The P2X₇ receptorexpression has been found in: i) normal tissues, Greig et al, 2003a; ii)precancerous epidermal tissues, Slater and Barden, 2005; and skin cancercells, Greig et al, 2003b; White et al, 2005; and Pastore et al, 2007.P2X₇ receptor immunoreactivity was found throughout the epidermis,including in the basal/parabasal germinative regions of the epidermis.Greig et al, 2003a. P2X₇ receptors were detected as early as 8-11 weeksin human fetal epidermis cells (i.e., for example, periderm), whereinthe receptors co-localized with caspase-3 and TUNEL staining. Greig etal, 2003c. Co-localization of the P2X₇ receptors with suchapoptosis-related markers was also reported in adult human epidermis,and recent studies reported BzATP-induced cell death in normal andcancer keratinocytes. Greig et al., 2003a, Greig et al, 2003b; andSlater & Barden, 2005.

Although these existing observational data suggest that the P2X₇ mayregulate growth of epithelial cells little is known about the biologicalrole of the P2X₇ receptor in the epidermal layers. For example, noprevious studies have investigated experimentally the biological role ofthe P2X₇ receptor in vivo. The data presented herein demonstrates thatP2X₇ receptors have an in vivo physiological role in the control ofgrowth of epidermal epithelial cells. The data also suggest that thisgrowth control may occur through apoptotic mechanisms, and thatpharmacological stimulation of the receptor could inhibit development ofepidermal neoplasia. For example, the presented data collected incultured human normal keratinocytes and/or cancer keratinocytes providedirect evidence that P2X₇ receptors control the growth of cells throughregulation of apoptosis. Specifically, in vivo mouse data discussedbelow show that locally applied P2X₇ receptor agonists inhibitDMBA/TPA-induced papilloma formation.

The translated product of the human P2X₇ transcript is a 595 aa linearpolypeptide, predicted to traverse the plasma membrane and to possesstwo intracellular domains and an extracellular domain with the followingtopology. See, FIG. 21A. The P2X₇ polypeptide may comprise the followingregions:

a) N-terminus (aa 1-25), which forms intracellular complexes withseveral proteins including β2 integrin, receptor-like tyrosinephosphatase (RPTP), α-actin, phosphatidylinositol 4-kinase,membrane-associated guanylate kinase, and several heat shock proteins[45]. These complexes may mediate some P2X₇-dependent signaling

b) The first transmembrane segment (aa 26-46)

c) Extracellular domain (aa 47-334), which contains the ligand bindingsite [46-49], and five putative N glycosylation sites [43], of whichAsn187, Asn213, and Asn241 are required to confer functionality [Ref.50; and Gorodeski, unpublished data]

d) The second transmembrane segment (aa 335-355).

e) A long C-terminus (aa 356-595) [43] that is required for poreformation [50,51]. Domains within the carboxy-terminal tail of the P2X₇also direct trafficking and stabilize expression of the receptor in theplasma membrane [50-54].

The functional P2X₇ receptor is a glycosylated G-coupled membrane-boundprotein. See, FIG. 21D. [41,42,47,48,55]. The natural ligand of the P2X₇receptor is ATP. See, FIG. 21B. [47,48]. ATP is present in extracellularfluids at high nanomolar, low micromolar concentrations [40,56-60],which suffice to activate the receptor [38,40-42,55]. Regulation of ATPin extracellular fluids was described [e.g. 46,61,62].

Activation of the P2X₇ receptor by ATP can stimulate various signalingpathways including the IL-1β [63], TNFα-TRAIL [64], and the p38,JNK/SAPK [65] and NF-kβ cascades [66], some of which can induceapoptosis. In epithelial cells, the quantitatively significantpro-apoptotic P2X₇ effect involves formation in the plasma membrane ofpores with a diameter of ˜4 nm that allow influx of molecules of 400-900daltons. See, FIG. 21E. [38,40-42,49,55]. In its maximal size, the poreis relatively permeable to Ca²⁺, but it remains selective to othercations and is impermeable to anions [49]. P2X₇ pore formation involvesrecruitment to the plasma membrane of receptor molecules [67-69] andpannexins [70,71], and the formation of complexes composed of receptortri-oligomers. See, FIG. 21D. [47-50]. P2X₇ molecules can also formstructural interactions with P2X₄ receptor molecules [73,74], which maystabilize the pores [75].

In epithelial cells e.g. human uterine ectocervical, endocervical andendometrial cells and human keratinocytes, activation of the P2X₇receptor induces pore formation and uncontrolled influx of Ca2+, whichtriggers mitochondrial damage followed by activation of caspase9/7/3-mediated apoptosis. See, FIG. 21F. [38,40-42,55].

Similar to other G-coupled protein receptors, activation stimulatesGRK-3-mediated phosphorylation of the P2X₇ receptor on tyrosine, serine,and threonine residues, and recruitment of β-arrestin-2 to the plasmamembrane [42]. β-arrestin binding facilitates uncoupling of the receptorfrom the heterotrimeric G proteins, and the activated phosphorylatedreceptor molecules participate in the stimulation of various signalingpathways [42]. However, most of the remaining activated receptormolecules are internalized by endocytosis, and 13-arrestin bindingtargets the receptor in a dynamin-related mechanism to clathrin-coatedpits. Endocytosis is followed by receptor sequestration into variouscellular domains, recycling, and degradation. See, FIG. 21D. [42].

A Biochemistry

P2X₇ is one of a number of pro-apoptotic systems that operate inepithelial tissues [37,38]. The P2X₇ receptor is expressed byproliferating cells [39], and activation of the receptor inducesapoptosis that controls directly the growth of the epithelial cells[38,40-42].

The human P2X₇ gene is localized within a 55 kb region of chromosome12q24; it has 13 exons that encode a 595 amino acid (aa) polypeptide[43]. The P2X₇ transcription initiation site (TpIS) is adenine (+1) atnucleotide (nt) 1683 of the human P2X₇ gene [GenBank Y12851]), with aTTAAA sequence at nt −32/−28 and an active promoter region within nt−158/+32 [44].

The low affinity of the P2X₇ receptor for ATP (EC₅₀ of 100 μM) and BzATP(EC₅₀ of 35 μM) [38,40,42,55] is unusual relative to the proposedphysiological significance of the P2X₇ receptor mechanism to apoptosis,since extracellular steady-state levels of ATP are only in the highnanomolar, low micromolar range. This question could be answered byreference to the biological role of the receptor. In epithelial cells,the P2X₇ receptor controls the growth of cells through the induction ofapoptosis [38]. A high affinity of the P2X₇ receptor for its naturalligand ATP would be disadvantageous to normal cells because the receptorwould be activated continuously and induce cell death. Cells haveapparently devised mechanisms to regulate expression and activity of theP2X₇ receptor, and the low affinity of the receptor could be anadditional physiologic mechanism to control receptor activation and toavoid cell death. See, FIG. 22. Under normal conditions, theextracellular levels of ATP are in the range that corresponds to thethreshold level for receptor activation [38,40,42,49,55]. Accordingly,only a fraction of the receptor pool would be activated, and only fewcells are likely to undergo apoptosis. This hypothesis is supported bythe mouse data in vivo that in the normal skin the degree of baselineapoptosis in P2X₇-receptor-expressing keratinocytes is low. See, FIGS.26C and 26L, [55].

B. Cellular Localization

The limiting factor of P2X₇-mediated apoptosis is cellular expression ofthe P2X₇ receptor [42,50]. Localization of the P2X₇ receptor has beenstudied by employing immune methods, and P2X₇ immunoreactivity was foundin different cellular locations. Co-localization in the plasma membranereflects foremost the full-length active receptor [42,50], althoughtruncated forms (e.g. the P2X_(7-j) protein) can be also sorted to theplasma membrane [50]. P2X₇ immunoreactivity found in the cytoplasmreflects newly formed receptor en route to be inserted into the plasmamembrane, or post-activation internalized, degraded, or recycledreceptor [50]. Perinuclear/nuclear P2X₇ immunoreactivity was describedin epithelial [76-78] and non-epithelial cells [79,80], but itsbiological significance is unclear.

In some types of epithelial tissues, e.g. the cervix and endometrium,the P2X₇ receptor is sorted predominantly into apical domains of theplasma membrane [81,82]. In the endometrium, apical expression of thereceptor depends on the level of tissue differentiation, and sortinginto apical regions of the plasma membrane is highest in tissues ofwomen at the late proliferative phase of the menstrual cycle [82]. Thebiological significance of these findings is unclear.

C. Regulation

One factor that determines activity of the P2X₇ system is the degree ofexpression in the plasma membrane of the full length functional receptor[42,50]. The P2X₇ system is a potent pro-apoptotic factor in vivo, andcells have developed mechanisms that regulate and control expression andfunction of the receptor [55].

1. Transcription Regulation

P2X₇ transcription is regulated by two groups of cis-regulatory enhancerelement(s) located within nt regions +222/+232 and +403/+573 downstreamof the active promoter. See, FIG. 22. [44]. Correlative data suggestthat these regions contain binding sites for transcription factors p300,Elk-1, E47, EIIaE, E2F, and p53 [44], which can integrate signals fromenhancer and promoter regions and regulate cell growth and apoptosis[83-90]. The repression or absence of some of these transcriptionfactors, e.g. E47 [85] and p53 [90], have been implicated in cancerdevelopment. The tumor suppressor p53 controls expression of genesinvolved in the regulation of cell cycle progression and cell death[90], and P2X₇ expression correlates with p53 protein levels [91], andwith activation of the p53 apoptotic pathway [92].

P2X₇ transcription is controlled by methylated cytosines atcytosine-phosphodiester-guanosines (CpG) sites that cluster orco-localize with the enhancers' sites. See, FIG. 22. [44]. Cytosines atCpG sites +211/+212 +330/+331 and +461/+464 are constitutivelymethylated in vivo, and hypermethylation of these sites inhibits P2X₇transcription [44]. Conversely, treatment of cells with de-methylationdrugs, e.g. 5-aza-2′-deoxycytidine (Aza-dC) upregulates P2X₇ mRNA,possibly by inducing de-methylation of cytosines at the CpG sites+211/+212 +330/+331 and +461/+464 [44]. The molecular mechanism by whichhypermethylated CpGs downstream of the P2X₇ promoter inhibittranscription possibly involves modulation of the spatial conformationof transcription factors recognition sites within the putative enhancersregions [44].

2. Post-Transcriptional Regulation

The full-length human P2X₇ 3′-untranslated region (3′UTR) containssequences that confer instability to the P2X₇ transcript [93,94]. Humanpoly(ADP-ribose) polymerase (PARP) interacts with transcribed P2X₇ mRNAand de-stabilizes the (3′UTR)-P2X₇ mRNA [93]. In contrast, inhibition ofPARP augments P2X₇-related apoptosis by increasing stability of the P2X₇mRNA. See, FIG. 22 [93].

The human P2X₇ 3′UTR also contains target sites for micro-RNAs (miRNA),which are small noncoding 18-25 nt RNAs that regulate mRNA targets [95].The human P2X₇ 3′UTR contains binding sites for miR-186 and miR-150,which confer instability to the P2X₇ transcript [94]. Overexpression ofmiR-186 and miR-150 inhibits synthesis of P2X₇ mRNA [94], whileinhibition of miR-186 and miR-150 upregulates synthesis of P2X₇ mRNA[94] and increases ligand-induced P2X₇ pro-apoptotic effects. See, FIG.22. [94, 96].

3. Regulation of Receptor Glycosylation

Membrane expression and functionality of the P2X₇ receptor depend onglycosylation of the receptor. Glycosylation facilitates Golgitransport, trafficking, and insertion of the receptor into the plasmamembrane, while receptor de-glycosylation abrogates receptor function[41]. Glycosylation of the P2X₇ receptor is controlled by(β2-adrenoceptor (β2-AR)—activation of protein kinase-A (PKA), resultingin de-glycosylation of the P2X₇ receptor and enhanced receptordegradation [41]. The PKA effect is regulated by the action of theepidermal growth factor (EGF); it involves facilitated, phosphoinositide3-kinase (PI3K)-dependent inhibition of β2-AR internalization, andfacilitated β2-AR recycling, thereby increasing the pool of β2-ARs inthe plasma membrane that are available for activation upon ligandbinding. See, FIG. 21D and FIG. 22. [41].

4. Regulation of Receptor Oligomerization

P2X₇-mediated apoptosis involves ligand-induced pore formation [47-49],which depends on homo(tri)-oligomerization of the full-length receptor.See, FIG. 21E. [47-50]. Oligomerization of the P2X₇ receptor depends onthe availability of P2X₇ monomers, and is influenced by the presence oftruncated forms of the P2X₇ that can hetero-oligomerize with thefull-length P2X₇ form and produce non-functional pores [50]. Fivetruncated variants of the human P2X₇ were previously reported(P2X_(7-b), P2X_(7-c), P2X_(7-g), P2X_(7-I), and P2X_(7-j)), resultingfrom alternative splicing [50,54,96]. All lack the carboxy terminus ofthe wild-type P2X₇ receptor, and some (P2X_(7-b) and P2X_(7-j)) havebeen shown to be ineffective in pore formation and apoptosis inductionwhen expressed singularly in host cells [50].

The P2X_(7-j) form is expressed naturally in normal and in cancerepithelial cells [50]. It is composed of the proximal 248 amino-acids ofthe wild-type P2X₇ with an altered stretch of 10 amino-acids at itscarboxy terminus. It lacks the distal 337 amino-acids of the P2X₇,including the entire intracellular carboxy terminus, the secondtransmembrane domain, and the distal third of the extracellular loop.When expressed heterologously, treatment with the P2X₇-specific ligandBzATP evokes minor channel activity but it fails to induce poreformation and apoptosis [50]. Co-expression in host cells of theP2X_(7-j) plus the full-length P2X₇ results in hetero-oligomerizationbetween the P2X_(7-j) and the P2X₇, and the formation of nonfunctionalP2X_(7-j)/P2X₇ hetero-oligomers [50].

Analysis of the oligomeric products in host cells co-expressing the P2X₇plus the P2X_(7-j) suggested formation of four types of trimericcomplexes in the following order of relative expression. See, FIG. 21E.[50]:

-   -   [P2X_(7-j)]³ (70%)    -   [P2X_(7-j)]²/[P2X₇] (15%)    -   [P2X₇]³ (10%)    -   [P2X₇]²/[P2X_(7-j)] (5%)

Of these, only the [P2X₇]³ forms a functional pore [50], indicating thatco-expression of the P2X₇ plus the P2X_(7-j) favors formation ofinactive complexes. The data suggest that in cells expressing bothreceptor forms, abundance of the P2X_(7-j) or paucity of the full-lengthP2X₇ receptor will increase the likelihood of formation of nonfunctionalpores, and will tend to abrogate the induction of apoptosis. See, FIG.22.

5. Estrogen Regulation of P2X₇-Mediated Apoptosis

In estrogen-responsive tissues, exposure to estrogen inducesgrowth-promoting effects on cells. In the past it was believed that mostof the effect involves activation of estrogen-dependent mitogenicstimuli. However, recent data show that some of the effect is the resultof estrogen induced anti apoptotic effect. Experiments using humannormal and cancerous estrogen-responsive uterine epithelial cells showedthat estrogen inhibits baseline apoptosis (mediated primarily by theP2X₇-receptor [38]), as well as P2X₇-augmented apoptosis [40]. In thesecells, the P2X₇-mediated apoptosis involves influx of Ca²⁺ viaP2X₇-pores and induction of apoptosis by the mitochondrial-caspase-9pathway [38, 40-42]. Treatment with estrogen blocksP2X₇-receptor-induced activation of caspase 9, but the mechanism ofapoptosis inhibition differs in normal and in cancer cells. In thenormal cells, treatment with estrogen results in lower cytosolic calciumby attenuation of P2X₇-induced calcium-influx. In contrast, estrogenup-regulates activity of the anti-apoptotic factor Bcl-2 in cancer cells[40].

D. Biological Effects

Activation of the P2X₇ receptor stimulates numerous effects [46-49]. Atthe tissue/organ level the P2X₇ is involved in the innate immuneresponse against microbial infections. Ligation of P2X₇ by ATP canstimulate inflammasome activation and secretion of proinflammatorycytokines such as IL-1β [e.g. 63, 97, 98], and the production ofreactive oxygen species (ROS). In macrophages, P2X₇ stimulates ROSproduction through the MAPKs ERK1/2 and the nicotinamide adeninedinucleotide phosphate oxidase complex [99]. P2X₇ effects also regulatethe function of the nervous [100], skeletal [101], and epithelialsystems [102]. At the cellular level, non-apoptotic effects of P2X₇receptor activation involve membrane responses, and alter profiles ofcell surface lipid and protein composition that modulate the directinteractions of P2X₇-receptor-expressing cells with other cell types.The responses can also induce the release of bioactive proteins, lipids,and large membrane complexes into extracellular compartments for remotecommunication between P2X₇-receptor-expressing cells and other cellsthat amplify or modulate inflammation, immunity, and responses to tissuedamage [103].

However, the hallmark of P2X₇ receptor activation isinduction/augmentation of cell death. Early studies reported on thecytolytic effects of P2X₇ receptor activation in cells of the whitelineage. Recent studies focused on the pro-apoptotic effects of P2X₇receptor activation in epithelial cells.

1. Normal Epithelial Cell-Growth

Data in normal human epithelial uterine cells (i.e., for example,ectocervical, endocervical or endometrial) and epidermal cells, and innormal mouse epidermal keratinocytes, suggest that the P2X₇ system playsa physiological role in the regulation of epithelial cell growth forreasons including, but not limited to:

-   -   a) The P2X₇ receptor is both necessary and sufficient for        controlling baseline apoptosis [38-42,50,55,94]    -   b) Blocking synthesis of the receptor abolishes baseline        apoptosis [38-42,50,55,94]    -   c) Baseline apoptosis is the result of activation of the P2X₇        receptor by ATP [38-42,50,55,94]    -   d) ATP, the natural ligand of the P2X₇ receptor, is present in        extracellular fluids at concentrations that suffice activation        of the receptor [38,40,55]    -   e) ATP is constitutively secreted by cells, acting in a        paracrine/autocrine manner [46-49]    -   f) Under normal physiological conditions other pro-apoptotic        systems e.g. the Fas and the TNF mechanisms play a minor role in        mediating baseline apoptosis [38]    -   g) In epithelial cells the P2X₇ receptor is expressed mainly in        reserve cells that determine the proliferative capacity of the        epithelium [39].

The expression profile of P2X₇ receptor in normal stratifying epitheliaexplains its fundamental role in the control of cell growth. Instratified epithelia, e.g. the uterine ectocervix [39,82] or theepidermis [55,76,105], cells proliferate from reserve cells that residein the basal/parabasal layers. These cells rest on the basal membraneand continue to divide throughout life. In the skin, a large pool ofreserve cells can be also found within hair shafts [105]. Superficial tothe parabasal layer are layers of metabolically active cells(intermediate in the cervix, granular in the skin), and layers of cellsundergoing terminal differentiation (superficial cells in the cervix,transitional cells in the skin). The skin, in contrast tonon-keratinizing stratified epithelia (e.g. the cervix) also containsthe superficial stratum corneum, composed of layer(s) of flat dead cells[105].

In normal stratifying epithelia, P2X₇ immunoreactivity is intense inbasal/parabasal layers (and in hair shafts in the skin); lesser in theintermediate ectocervical layers of the epithelium and in the epidermalgranular layers; and more intense in the superficial (ectocervix), andtransitional and stratum corneum layers (skin) [39,76,82,104]. Incontrast to the P2X₇ receptor protein, P2X₇ mRNA is found mainly in thebasal/parabasal layers, but not in the more superficial layers [39].Thus, the P2X₇ immunoreactivity in non-proliferating layers representsreceptor trapped in dying cells that undergo terminal differentiation,and most likely it mediates the apoptosis that is associated with theterminal differentiation [105]. However, this function is secondary toits main role, which is regulation of growth of the proliferatingreserve cells. Reserve cells are the target of carcinogenic stimuli andare at risk of undergoing neoplastic transformation; the intenseexpression of de-novo synthesized receptor in these cells suggests thatthe P2X₇ receptor controls directly the growth of the reserve cells[55].

2. Cancer Epithelial Cell-Growth

While P2X₇ receptor activation may augment apoptosis in both normal andcancer epithelial cells, some types of cancerous epithelial cells have adecreased apoptotic effect than in the respective normal cells. Forinstance, treatment with the P2X₇ receptor specific agonist BzATP ofcancer ectocervical, endocervical, endometrial cells and cancerkeratinocytes resulted in 2-5 fold lesser apoptosis than in therespective normal cells [38,40,55]. Baseline apoptosis, which in thosecells is determined mainly by the P2X₇ system [38,40,55], was alsosmaller in the cancer cells than in the normal cells [38,40,55]. Recentdata indicate that the attenuated P2X₇-mediated apoptosis in the cancercells is due mainly to reduced cellular levels of the P2X₇ receptor(mRNA and protein) in the cancer cells [41,42,44,50,55,94].

Differences in P2X₇ receptor expression in cancer versus normalepithelial cells were described [82]. Recent studies usingwell-characterized immune methods to detect the functional full-lengthP2X₇ receptor reported that epithelia can be grouped relative to P2X₇receptor expression as follows:

-   -   a) Epithelia with similar trend of expression in normal and        cancer cells, e.g. the colon [82].    -   b) Epithelia with higher expression of the P2X₇ in cancer cells        than in normal cells, e.g. thyroid [82,106].    -   c) Epithelia with lesser expression of the P2X₇ in cancer cells        than in normal cells. These include epithelia derived from the        ectoderm (skin and breast), the uro-genital sinus (bladder and        ectocervix), and the distal paramesonephric (Müllerian) duct        (endocervix and endometrium) [39,50,55,76,82,104]. In those        tissues P2X₇ expression is decreased already in early phases of        the neoplasia.

Thus, early embryonic events may imprint the type of P2X₇ regulation ofexpression in later stages of life.

P2X₇ polymorphisms have been described and point-mutations have beenidentified in lymphocytes and monocytes, causing eitherloss-of-function, e.g. 1513A>C (E496A), 1729T>A (1568N), 946G>A (R307Q),1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R); or gain offunction, e.g. 489C>T (H155Y). A 1352T>C(P451L) change impairs celldeath in murine thymocytes. Polymorphisms in the promoter region of theP2X₇ receptor has also been reported, and one variant was associatedwith protection against tuberculosis. Additional P2X₇ gene variants wereincluded in the Single Nucleotide Polymorphism Consortium database, buttheir functional implications remain to be determined [50]. Numerouspoint-mutations were found in cancer epithelial cells, [data not shown],but there is no evidence that genetic mutations of P2X₇ influence thedevelopment of cancer in these tissues.

In contrast to genetic mutations, studies found that differences in theepigenetic control of transcription, mRNA stability, glycosylation, andoligomerization can explain the attenuated expression of the functionalP2X₇ receptor in the cancer epithelial cells, for example:

Hypermethylation

Hypermethylation of genes can induce repression of tumor suppressorgenes and lead to cancer development [107]. In uterine cervical cellsCpG sites +193/+194 (and/or +211/+212), +330/+331, and +461/+462 (and/or+463+464), in regions that co-localize with enhancers' sites, aredirection-dependent inhibitory cis elements of P2X₇ transcription [44].Data in cultured cervical cells and in cervix epithelial tissues in vivoshowed that cytosines within these CpG sites are hypermethylated in thecancer cells [44]. Moreover, the hypermethylation of cytosines in CpGsites downstream of the P2X₇ promoter correlated with reduced expressionof P2X₇ mRNA and protein in the cancer cervical cells [44], suggestingthat decreased transcription of P2X₇ through hypermethylation ofcis-regulatory elements plays a role in cancer development.

Steady State Levels

Low steady state levels of P2X₇ in cancer epithelial cells can be theresult of enhanced degradation of the transcript through the action ofmicro-RNAs [94] miRNAs reside in genomic regions that are involved inthe regulation of cell growth, and abnormally expressed miRNAs in humancancers can modulate the stability of transcripts of protein-codinggenes involved in tumorigenesis or apoptosis [94]. Sequences within thehuman 3′UTR-P2X₇ express target sites for miR-186 and miR-150 thatconfer instability to the P2X₇ transcript [94]. Cancer epithelial cellsexpress higher levels of micro-RNAs miR-186 and miR-150 than normalcells [94], and the increased expression of miR-186 and miR-150stimulates degradation of the P2X₇ transcript preferentially in cancercells.

Glycosylation

Glycosylation of the P2X₇ receptor is important for receptor function.In epithelial cells glycosylation of the P2X₇ receptor is controlled bythe EGFR/PI3K/β2-AR/PKA systems, and activation of PKA by the β2-ARmechanism induces de-glycosylation of the P2X₇ receptor [41]. The β2-AReffect is facilitated by co-activation of the EGFR. which is oftenoverexpressed in cancer epithelial cells. See, FIGS. 21D and 22, [41]and [108], respectively. Therefore, overexpression of the EGFR couldcontribute to neoplasia by two groups of mechanisms: via itspro-mitogenic influence; and by abrogation of P2X₇-mediated apoptosis(reduced expression of the P2X₇ receptor through PKA-mediatedde-glycosylation of the receptor).

Splicing Variants

Nine splice variants of the human P2X₇ resulting from alternativesplicing were previously reported, of which the P2X_(7-j) is the mostprevalent in epithelial cells. The P2X_(7-j) can hetero-oligomerize withthe full-length receptor, and lead to the formation of inactivecomplexes. In contrast to the full length P2X₇, which is reduced in sometypes of cancer cells, the expression of the P2X_(7-j) in those cells isrelatively stable. This imbalance would favor the formation of inactiveP2X_(7-j)/P2X₇ hetero-oligomers leading to defective apoptosis andaugmented growth of the cancer cells. See, FIG. 21E, [50].

III. Cancer Treatment

Some reports suggest that pro-apoptotic activity mediated by the P2X₇receptor could be potentially used as a cancer treatment and cancerprevention in humans [110]. Until the present invention, however, thedevelopment of such an anti-neoplastic modality was not clinicallyrelevant because target tissue specificity and an understanding of thepharmacological properties of an effective therapeutic was notunderstood.

It is currently believed that a P2X₇ receptor is member of the family ofATP-dependent P2 nucleotide purinergic receptors. Early data on theanti-neoplastic properties of P2 receptors agonists prompted in vivostudies using systemic or intraperitoneal treatments with ATP, thenatural ligand of P2 receptors. [111]

Cancer treatments using intravenous infusions of ATP in patients withadvanced cancers have been reported. For example, a randomized clinicaltrial in patients with advanced non-small-cell lung cancer has shownbeneficial effects of ATP treatment on fatigue, appetite, body weight,muscle strength, functional status, quality of life, and serum albuminconcentrations, with marked effects in cachectic patients [112]. ATPtreatment was associated with increased survival in the subgroup ofweight-losing patients (9.3 months in ATP-treated patients versus 3.5months in control patients); however, ATP had no effect on tumor stageand survival within the entire study group [113]. The data suggestedthat the beneficial effects of ATP on body fat, fat-free and body cellmass, and on energy intake were due to the maintenance of energy intakeby restoration of hepatic energy levels. [114, [115], respectively.

Although the above studies suggest that palliative care of terminallyill cancer patients might be useful using intravenous ATP, it is evidentin retrospect that the use of systemic ATP as an-anti cancer modalitywas likely to fail. Systemic administration of ATP will target P2receptors throughout the body. Cells express more than one type of P2receptors, and responses to ATP depend on the type of P2 receptors thatare activated. Also, depending on cell type, activation of some receptorsubtypes (i.e., for example, P2Y₁, P2X₅, and P2X₇) leads to a decreasein cell number, whereas activation of other receptor subtypes (i.e., forexample, P2Y₂) can lead to an increase in cell number [116]. Inaddition, in some cell types e.g. peripheral T lymphocytes, activationof the P2X₇ receptor can produce mitogenic response [117].

ATP instability is another factor that could negatively affect ATP'sefficacy such as by ectoATPase degradation in extracellular fluids[118]. For example, anti-cachectic effects could have occurred by abreakdown product of ATP, adenosine, which is effective at reducingweight loss [119]. Ecto-ATPases are known in the plasma andextracellular fluids [61]. Further, it is unlikely that ATP reached aconcentration range that is required for pharmacological activation ofthe P2X₇ receptor [38,40,42,55].

Thus, the lack of tissue specificity and the poor understanding of ATPpharmacology following the systemic administration of ATP at the time ofthese studies provide a rationale for their clinical failure.

Recent studies have examined the effects of intraperitonealadministration of ATP on tumors resulting from implanted prostate andbladder carcinomas cells in nude mice [120], [121], respectively. Thedata showed a 44-69% reduction in the growth of freshly implanted cancercells, with no adverse effects on the host mice. The growth reduction ofthe implanted bladder and prostate cancers provides proof-of-concept forthe anti-neoplastic effect of ATP. [119]. Based on these considerations,the art suggests that the use of ATP as an anti-neoplastic modalitycould produce non-specific responses, and that ATP should not be used asan anti-neoplastic treatment modality in humans. Instead, one shouldfocus on the use of P2 receptor modulators (i.e., for example, agonistsor antagonists) that are selective for mediating the growth of cancercells [116].

In one embodiment, the present invention contemplates a compositioncomprising BzATP as one such ATP agonist. BzATP was used to activateP2X₇-mediated apoptosis and modulate growth of epidermal neoplasia. [55}BzATP was synthesized and used initially as a photoactivatable,covalently binding affinity probe to study site-specific adeninenucleotide binding to the ATPase of submitochondrial particles [122]. Incontrast to ATP which behaves in aqueous solutions as a weak acid(ATP⁴⁻), BzATP has lipophilic characteristics that are contributed bythe benzoyl-benzoyl ring, and which could explain its penetrationthrough the epidermis. See, FIG. 21C. BzATP is a relatively stablecompound in biological solutions and across a broad range oftemperatures and pH, although serum albumin and various plasma lipidscan bind BzATP and reduce its potency at P2X₇ receptors [123]. BzATP iscommercially available in the form of either2′(3′)-O-(4-benzoyl-benzoyl)adenosine-5′-triphosphate (C₂₄H₂₄N₅O₁₅P₃,MW=715.39) or the tri-triethylammonium salt, C₂₄H₂₄N₅O₁₅P₃.C₁₈H₄₅N₃,MW=1018.97). Both BzATP forms have been shown to exert similaranti-apoptotic effects. (data not shown)

BzATP is a potent and efficacious ligand of the P2X₇ receptor, with a5-10 fold greater potency than ATP [42,50]. Activation of the P2X₇receptor requires the continued presence of the ligand, and treatmentwith BzATP exerts a prolonged P2X₇ effect [124]. In most studied celltypes BzATP has been showed to be a specific P2X₇ receptor agonist[46-48], but in some cell types BzATP can act as a weak agonist, or aweak antagonist, depending upon the particular P2 receptor subtype: i)P2Y_(1 [)125-127]; ii) P2Y2 [128], and iii) P2X₁, P2X₂, and P2X₃receptors [48,129]. A study in hippocampal mossy fibers suggested thatBzATP can be catabolized extracellularly by ecto-nucleotidases toBz-adenosine, which can then be transported intracellularly vianucleoside transporters. [130]. Once transported intracellularly, thebenzoyl-benzoyl group can be removed by intracellular esterases and theadenosine is released into the extracellular space thereby activatingadenosine receptors.

Despite those isolated reports, most studies using BzATP concur that thepredominant observed effects are the result of activation of the P2X₇receptor [131]. Various epithelial cell types, including human androdents' keratinocytes, have indicated that BzATP effects are notmediated by degradation products of BzATP (e.g. Bz-adenosine5′-diphosphate ADP [BzADP], adenosine 5′-monophosphate [AMP], oradenine). In addition, treatments with adenosine 5′-diphosphate (ADP),AMP, adenosine, or adenine had no pro-apoptotic effects on the cells.(data not shown).

The data presented herein demonstrates that BzATP applied locally on theskin, at areas at risk for developing skin papillomas and cancers,showed 50% inhibition of papillomas and skin cancers in animals thatwere treated with BzATP. It is further shown that a main target of BzATPtreatment are P2X₇-receptor-expressing reserve cells. In the normalskin, BzATP augmented apoptosis of the reserve cells without evokinginflammatory or atrophic changes, which potentially could have beeninduced by activation of the P2X₇ receptor [46-49]. Specifically, BzATPdid not induce tissue loss, thinning, or ulceration. In contrast,treatment with BzATP inhibited papilloma formation, and it inhibited thetransformation of papillomas to skin cancers. In the DMBA/TPA mousemodel, most early papillomas are histologically benign, but some willprogress to squamous cell carcinoma [132,133]. Papillomas at risk fordeveloping into cancer are characterized by increased proliferativecapacity of epidermal reserve cells [132]. P2X₇-mediated apoptosis haslittle effect on slow growing normal cells, but it controls theproliferation of rapidly growing cells, while BzATP has the potentialfor skin chemoprevention by controlling the development of precancerouscells and their transformation into skin cancers. [55] BzATP alsoinduced apoptosis of advanced cancer cells in vivo, but the effect waslimited by the low expression of the P2X₇ receptor in these cells. See,FIGS. 26T,26V and FIGS. 26S,26U, respectively. P2X₇ receptor levels canbe increased in normal and in cancer cells by inducing de-methylation ofcytosines at CpG sites that control P2X₇ transcription. See, FIG. 22;[44].

Collectively, these data suggest that the pharmacological augmentationof P2X₇-mediated apoptosis are prophylatic for skin cancers, andtherapeutic for early skin cancerous lesions.

The initial studies utilized a low BzATP dose of the BzATP and did notresult in any local or systemic adverse events. This implies that whenadministered locally on the skin, BzATP may be a candidatechemotherapeutic growth-preventive drug with high therapeutic and lowtoxic pharmacological profile. Other epithelia could be considered aspotential targets for local treatment with BzATP as well, including theoral mucosa, the vulvar and vaginal epithelia, and the ectocervix. Thesesquamous stratified epithelia are phenotypically similar to the skin;their reserve cells express the P2X₇ receptor, and activation of thereceptor induces apoptosis. Cancers of these tissues usually developfrom premalignant lesions, and similar to the skin the cancer risk canvary from 0.1% to 20% [134-136]. Although it is not necessary tounderstand the mechanism of an invention, it is believed that activationof P2X₇-dependent apoptosis with BzATP could be a novel chemotherapeuticgrowth-preventive modality for pre-cancerous and early cancerousepithelial lesions.

In one embodiment, the present invention contemplates a method fortreating and preventing cancer comprising activating a P2X₇ receptor.For example, the data presented herein show that P2X₇-mediated apoptosiscontrols the growth of skin epithelial cells. In particular, a localapplication of a P2X₇-specific agonist ligand (i.e., for example, BzATP)inhibits formation of DMBA/TPA-induced papillomas in mice in vivo.Although it is not necessary to understand the mechanism of aninvention, it is believed that the observed inhibition may be mediatedby a mechanism associated with activation of P2X₇-induced apoptosis.

The data presented herein also show that a P2X₇ receptor is bothnecessary and sufficient to mediate the BzATP pro-apoptotic effect. ATPconcentrations in the high nanomolar range of conditioned media of bothnormal human keratinocytes and human SCC-9 cells support the suggeststhat a P2X₇ receptor mechanism may play a role in in vivo cellregulation of apoptosis and growth. Accordingly, ATP may be secreted bycells into the extracellular milieu, wherein the resultant steady-stateATP levels are sufficient to activate the P2X₇ receptor and induceapoptosis. Since steady-state extracellular levels of ATP are similarbetween normal keratinocytes and SCC-9 cells (infra), it is suspectedthat P2X₇-mediated apoptosis may depend primarily on the expression ofthe receptor. (Wang et al, 2004a; Feng et al, 2005). In other types ofepithelial cells, a lower cellular expression of P2X₇ mRNA and receptorlevels appear in cancer cells versus normal epithelial cells. Thisdecreased expression was associated with a decreased P2X₇-mediatedbaseline apoptosis rate and/or ligand-induced apoptosis in cancer cellsas compared to normal cells. (Wang et al, 2004a; Li et al, 2006; Li etal, 2007). These data may be biologically and clinically importantbecause defective apoptosis could lead to cancer. Gasser and Raulet,2006; Kujoth et al, 2006; Rodriguez-Nieto and Zhivotovsky, 2006.Further, decreased cellular expression of P2X₇ receptor could becausally related to the development of epithelial cancers.

In one embodiment, the present invention contemplates a method formodulating the growth of epidermal cells by controlling P2X₇-mediatedapoptosis. In one embodiment, the epidermal cells comprise germinativeepidermal cells. In one embodiment, the controlling comprises drugswhich activate the P2X₇ receptor, wherein formation of a skin neoplasiais inhibited.

A. Anti-Cancer Agent Screening

Potential effective drugs may be screened by using a DMBA/TPA mouse skinchemical carcinogenesis model system that is capable of inducing skinsquamous cell carcinoma within 20 weeks of treatment. In the course ofcancer development, skin papillomas usually arise first (i.e., within6-15 weeks after initial DMBA/TPA exposure. Glick et al, 2007. The datadiscussed below using this model showed for the first time thatpharmacological stimulation of the P2X₇ receptor can inhibit developmentof epidermal papillomas. Specifically, when DMBA/TPA-treated mice wereco-treated with BzATP the incidence of papilloma formation wasdecreased. In one embodiment, the mean number of papillomas per animaldecreased by about 25%. In one embodiment, the mean papilloma sizedecreased by about 45%. In one embodiment, treated papillomas involutedmore frequently. Although it is not necessary to understand themechanism of an invention, it is believed that a pharmacologicalactivation of the P2X₇ receptor in vivo can inhibit formation ofDMBA/TPA-induced skin papillomas and stimulate involution of formedpapillomas.

It is further believed that BzATP treatment effects do not induce anyinflammatory skin changes that could potentially be related to P2X₇actions. Dubyak and el-Moatassim, 1993; Ralevic and Burnstock, 1998.Also, BzATP treatment did not produce other gross or microscopic effectson the skin, and there were no abnormal changes that could be the resultof excessive apoptosis such as thinning or ulcerations. It should benoted that Ki67 levels did not change during BzATP treatment, therebysupporting the conclusion that BaATP effects are primarily due to P2X₇receptor mediated effects.

Until the present invention, the mechanism by which BzATP-augmentedP2X₇-apoptosis inhibited DMBA/TPA-induced papilloma formation wasunknown. While it is generally believed that carcinogens (e.g. DMBA/TPA)may stimulate uncontrolled growth of proliferating (germinative) cells,the underlying biochemical control of cell growth may be controlled byP2X₇-mediated apoptosis (infra). It is therefore possible that thegrowth and development of papillomas induced by DMBA/TPA was balanced bythe apoptotic activity of BzATP-P2X₇. One possibility is that these twoeffects are independent of each other and that the development and rateof growth of papillomas depend on the contributions to regulation ofcellular growth made by these two opposing stimuli. An alternativehypothesis, is that activation of the P2X₇ receptor inhibits DMBA/TPAcellular and molecular proliferative effects. The signaling pathway andmolecular mechanism by which activation of the P2X₇ receptor inducesapoptosis in keratinocytes has not been clearly characterized.

In one embodiment, the present invention contemplates a method fortreating cancer comprising at least a partial inhibition of papillomaformation. In one embodiment, the inhibition is a result of treatmentwith a P2X₇ receptor agonist. In one embodiment, the P2X₇ agonistcomprises BzATP. In one embodiment, the agonist is provided as a localapplication. In one embodiment, the local application comprises aconcentration of approximately 1 μg/cm² BzATP. Although it is notnecessary to understand the mechanism of an invention, it is believedthat these inhibitory effects are dose-related such that higher doseshave a greater effect in inhibiting papilloma formation.

In the DMBA/TPA mouse model, most early papillomas are histologicallybenign, but some progress to a squamous cell carcinoma stage. Glick etal, 2007. The potential for malignant conversion in this model islargely cell autonomous, independent of microenvironmental influences.Woodworth et al, 2004. Papillomas at risk for developing into cancer areusually characterized by increased proliferation of keratinocytes in thebasal and suprabasal layers. Glick et al, 2007. This characteristic isrelevant because (a) the P2X₇ receptor is expressed in basal/parabasallayers of normal stratifying epithelia (Li et al, 2006) including theepidermis (Greig et al, 2003a; and the data presented herein); (b)decreased expression of the P2X₇ receptor in basal/parabasal layers canbe identified already in premalignant lesions of stratifying epithelia(Li et al, 2006); (c) treatment with BzATP induced activation of theP2X₇ receptor in basal/parabasal layers (data presented herein); and (d)BzATP treatment stimulated apoptosis also in basal/parabasal layers ofkeratinocytes outgrowths at the base of already developed papillomas.See, FIG. 5B(d). Therefore, BzATP may have both preventive andtherapeutic effects on the growth of papillomas.

In one embodiment, the present invention contemplates a method fortreating human epithelial cancers. In one embodiment, a P2X₇ receptor isexpressed by most types of epithelial cells. Surprenant et al, 1996;Ralevic and Burnstock, 1998; Khakh et al, 2001; Wang et al, 2004b; Fenget al, 2006; Li et al, 2006; Li et al, 2007. It is believed thatepithelial cancers usually develop from premalignant lesions, anddecreased expression of the P2X₇ receptor occurs already in earlypremalignant lesions. Li et al, 2006; Li et al, 2007. Cancer risks ofpremalignant lesions of stratifying epithelia, e.g. the oral mucosa(leukoplakia), cervix (dysplasia) and the epidermis (actinic keratosis)may vary form 0.1% to 20%, but at present it is difficult to predictwhich lesions will progress into cancer. Reibel, 2003; Fu and Cockerell,2003; Lindeque, 2005.

In one embodiment, the present invention contemplates a compositioncomprising at least one compound capable of activating a P2X₇ receptor,wherein the growth and progression of epithelial lesions are inhibitedin individuals at risk. In one embodiment, the compound comprises BzATP.The data described herein show that BzATP can be absorbed through theskin and may specifically target cells in the germinative layers of theepithelium at the site of drug application. Although it is not necessaryto understand the mechanism of an invention, it is believed thatpharmacological activation of the P2X₇ receptor may control the growthand progression of premalignant lesions through apoptosis withoutevoking necrosis and an inflammatory or immune response.

B. Intracellular P2X₇ Expression

In one embodiment, the present invention contemplates a method fortreating cancer comprising increasing P2X₇ receptor gene expression.Although it is not necessary to understand the mechanism of aninvention, it is believed that the reduced pro-apoptotic effect of BzATPin mouse cancer keratinocytes is possibly the result of low expressionof the P2X₇ receptor.

1. Normal Cells

The data presented herein confirmed that P2X₇ receptor is expressed inepidermal cells. In situ hybridization assays in cultured human normalkeratinocytes detected binding of an anti-sense full length P2X₇ cDNAprobe in perinuclear/cytoplasmic regions, with no reaction to the senseP2X₇ cDNA probe. See, FIG. 1A and FIG. 1B, respectively. Similarly, incultured human normal keratinocytes, P2X₇ immunoreactivity was found inthe cytoplasm and in the plasma membrane. See, FIG. 1C.

2. Human Epidermal Squamous Cell Carcinoma-9 Cells

Previous studies in uterine epithelia showed that P2X₇ mRNA and P2X₇receptor levels are lower in cancer cells as compared to correspondingnormal epithelial cells. Li et al, 2006; Li et al, 2007. The datapresented herein show similar trends in epidermal cells. See, FIGS. 1Cand 1D. P2X₇ immunoreactivity was 3 fold lower in SCC-9 cells than innormal keratinocytes. See, FIGS. 1C and 1D. Likewise, P2X₇ mRNA levelswere 6 fold lower in SCC-9 cells than in normal keratinocytes. See, FIG.1E. The decreased P2X₇ receptor expression correlated with decreasedapoptosis in these cells. For example, both baseline apoptosis andATP-induced apoptosis were lower in SCC-9 cells than in normalkeratinocytes. See, FIG. 1E, insert.

3. Mouse Skin Cancer Cells

Cellular effects of BzATP are believed to be mediated by the P2X₇receptor. Dubyak et al., “Signal transduction via P2-purinergicreceptors for extracellular ATP and other nucleotides” Am J Physiol1993, 265:C577-C606; and Ralevic et al., “Receptors for purines andpyrimidines” Pharmacol Rev 1998, 50:413-492.

The data presented herein show that normal cells, papilloma cells, andcancer skin cells in the mouse express the P2X₇ receptor, but cancerskin cell levels were significantly lower than either normal skin cellsor papilloma cells. See, FIG. 12. Immunostaining with the anti-P2X₇receptor antibody of tissue cross sections containing normal skinrevealed intense immunoreactivity that localized predominantly in theepidermis within proliferating keratinocytes and epidermal hair shafts.See, FIGS. 12A and 12B.

In papillomas, P2X₇ immunoreactivity was intense, similar to normaltissues, and it localized predominantly within proliferatingkeratinocytes at the base of the developing papillomas. See, FIGS. 12Cand 12D, respectively. In contrast, P2X₇ immunoreactivity in cancertissues was significantly lower than in normal epidermal or papillomatissues. See, FIGS. 12E and 12F, respectively. However, P2X₇immunoreactivity was four-fold less in cancer than in normal tissues.See, FIG. 12G.

This immunostaining data were confirmed by Western blot experiments.Quantitative assay of a P2X₇-specific band (i.e., for example, 75 KDa)revealed a five-fold lower density in cancer tissues than in normaltissues. See, FIG. 12H. Further confirmation was obtained by P2X₇ mRNAexperiments where quantitative polymerase chain reaction (qPCR) assaysrevealed a five-fold lower P2X₇ mRNA/GAPDH mRNA ratio in normal tissuesthan in cancer tissues. See, FIG. 12I. Collectively, these data indicatethat P2X₇ receptor expression levels in mouse skin cancer tissues arefour-five fold lower than in mouse normal skin tissues.

The effects of BzATP on P2X₇ expression and apoptosis (determined byTUNEL) in was determined in art accepted tissue models for papilloma andcancer tissues. In these studies, P2X₇ immunoreactivities in crosssections of DMBA/TPA-induced papillomas did not differ from normal cellsin either baseline intensity or after BzATP treatment. See, FIG. 15A andFIG. 15B. In contrast, however, the baseline intensity of P2X₇immunoreactivity in cross sections of skin cancers was significantlylower than in normal and in papilloma tissues. See, FIG. 12A and FIGS.15A & 15B, respectively. However, as with the papillomas, treatment withBzATP did not effect P2X₇ receptor expression in cancer cells. See,FIGS. 15E and 15F, respectively.

TUNEL staining was weak in cross sections of papillomas and cancertissues from the DMBA/TPA group. See, FIGS. 15C and 15G, respectively.Similar findings were observed in cross sections of normal skin. See,FIGS. 13F and 14G. In contrast, TUNEL staining was more intense in crosssections of papillomas and cancer tissues from the DMBA/TPA+BzATP group.See, FIG. 15D and FIG. 15H. In papillomas obtained from mice of theDMBA/TPA+BzATP group, enhanced TUNEL staining decorated basal/parabasallayers of keratinocytes outgrowing at the base of the developingpapilloma. See, FIG. 15D. These data suggest that although BzATP doesnot effect P2X₇ receptor expression, P2X₇-mediated apoptosis is,nevertheless, increased.

Morphologically, BzATP delayed formation of DMBA/TPA-induced papillomas,and resulted in fewer and smaller papillomas. Although it is notnecessary to understand the mechanism of an invention, it is believedthat some papillomas may have spontaneously regressed and involuted.Glick et al., “The high-risk benign tumor: evidence from the two stageskin cancer model and relevance for human cancer” Mol Carcinogenesis2007, 46:605-610.

However, the majority (i.e., for example, about two thirds) ofpapillomas either progressed into squamous spindle-cell carcinomas orpersisted as non-cancerous lesions. Persistence as non-cancerous lesionswas increased after BzATP treatment. For example, in mice co-treatedwith BzATP the proportion of animals with cancers at week 14 was lowerthan in the DMBA/TPA+BzATP group (50% versus 80%) and remainedrelatively stable. In contrast, the proportion of animals with cancersincreased steadily in the DMBA/TPA group, reaching 100% at week 24.These data suggest that local treatment with BzATP inhibits formation ofDMBA/TPA-induced skin papilloma, and it can also inhibit papilloma celltransformation into cancer cells.

BzATP had little effect on the number of cancerous lesions per animal atweeks 14-28, and on the proportion of animals with cancerous lesions >10mm³ at weeks 14-22. In contrast, after week 23 the proportion of livinganimals with cancerous lesions >10 mm³ increased in the DMBA/TPA groupwhile it had decreased in the DMBA/TPA+BzATP group. These data suggestthat local treatment with BzATP exerts an inhibitory effect on thedevelopment on skin neoplasia.

Interestingly, at weeks 15-24, among animals with cancerous lesions, theproportion of living animals with lesions larger than 200 mm³ tended tobe higher in the DMBA/TPA+BzATP group than in the DMBA/TPA group. Thiseffect cannot be explained by augmented proliferation since BzATP didnot stimulate DNA synthesis in cultured normal keratinocytes. Instead,the effect could be explained by comparing the survival curves (See,FIG. 11B) and the proportions of animals with smaller and larger sizecancerous lesions. See, FIGS. 10B & 10C. Thus, cancer-related deaths inthe DMBA/TPA group were associated more often with smaller lesions whilecancer-related deaths in the DMBA/TPA+BzATP group were associated withrelatively larger lesions. This suggests that treatment with BzATP alsoprolonged the life of animals with developed cancers.

B. P2X₇-Mediated Cell Apoptosis

In one embodiment, the present invention contemplates a method fortreating cancer comprising inducing P2X₇-mediated cellular apoptosis.Although it is not necessary to understand the mechanism of aninvention, it is believed that P2X₇-dependent apoptosis plays a role incontrolling the development and progression of cancers including, butnot limited to, epidermal neoplasias.

It was previously suggested that the P2X₇ mechanism controls growth ofepithelial cells through activation and modulation of apoptosis, Wang etal, 2004a. To better understand the cellular effects of BzATP in vivo,experiments investigated the effects of BzATP in the normal mouse onskin morphology and histology, immunoreactivities of P2X₇ antibody, andapoptosis.

1. In Vitro Effects of Exogenous BzATP

First, the apoptosis dose-requirements of ATP and of the P2X₇-specificagonist BzATP were determined. Treatment of keratinocytes and SCC-9cells with ATP, or with the P2X₇-specific agonist BzATP, augmentedapoptosis in a dose-related manner. See, FIG. 2A. It can be observedthat ATP was effective at inducing apoptosis at a 100 nM concentration.The data also showed that the combined pro-apoptotic effects of BzATP(100 μM) with ATP, at either 1 μM or 250 μM were non-additive. Incontrast, induction of apoptosis with TNFα in combination with BzATP wasadditive. See, FIG. 2A, Insert a). These data suggest that ATP, but notTNFα, induce apoptosis via the same mechanism of BzATP, i.e. the P2X₇receptor mechanism. Intracellular ATP levels were observed to be 610±50nM and 460±250 nM, respectively. These data indicate that ATP ismaintained within a concentration range sufficient to activateP2X₇-mediated apoptosis. See, FIG. 2A, Insert b.

It has been suggested that keratinocytes express different types ofpurinergic receptors and that it might be possible that, in addition tothe P2X₇ receptor, ATP-related apoptosis was mediated by other types ofpurinergic receptors. Burnstock, 2006. The above data does not supportthis speculation. First, the ATP concentration threshold for inducingapoptosis is consistent with a P2X₇-mediated effect. Dubyak andel-Moatassim, 1993; and Ralevic and Burnstock, 1998. Second, thecombined pro-apoptotic effects of a low (1 μM) or a high concentrationof ATP (250 μM) with a high concentration of BzATP (100 μM) did notproduce additive responses, suggesting that ATP and BzATP act via acommon mechanism (presumably the P2X₇ receptor). In contrast, thecombined pro-apoptotic effects of BzATP and TNFα did produce an additiveresponse, suggesting that BzATP and TNFα act via different mechanisms(i.e., for example, the P2X₇ receptor versus the TNFα-TRAIL pathway,respectively). Aggarwal and Rath, 1999. Third, the present data ruledout the possibility that the ATP/BzATP pro-apoptotic response wasnon-additive due to limited capacity of the cells to undergo apoptosis,because the pro-apoptotic effect of BzATP plus TNFα was greater than theBzATP only effect.

2. In Vitro Recombinant P2X₇ Expression

MDCK cells heterologously expressing the human full-length P2X₇ receptortagged with N-Myc at the N-terminus (N-Myc-hP2X₇-MDCK) have been used tostudy the P2X₇ receptor pathway. MDCK are epithelial cells that lackendogenous expression of the P2X₇ receptor. Feng et al, 2006; and Li etal, 2006.

Laser-confocal microscopy of N-Myc-hP2X₇-MDCK cells showedP2X₇-immunoreactivity decorating the plasma membrane. See, FIG. 2B,Insert: Panels a and b. Western blots of N-Myc-hP2X₇-MDCK cells lysatesrevealed co-immunoreactivity of a 75 kDa form with anti-Myc andanti-P2X₇ antibodies. See, FIG. 2B, Insert: Panels c-e. These data areconsistent with earlier reports suggesting expression of a functionalP2X₇ receptor. Feng et al, 2006. Confirmation of such expression wasdetermined by treatment of N-Myc-hP2X₇-MDCK cells with anti-sense P2X₇oligonucleotides that inhibited expression of the 75 kDa P2X₇ receptor.Treatment with random-control P2X₇ oligonucleotides had no such effect.See, FIG. 2B, Insert: Panels c and d).

To test the effect of heterologous expression of the human full-lengthP2X₇ receptor in MDCK cells on baseline and agonist-induced apoptosis,N-Myc-hP2X₇-MDCK cells were treated with BzATP. Wild-type (WT) MDCKcells, which lack endogenous expression of P2X₇, were used as a controlcell line. Feng et al, 2006. In WT-MDCK cells, treatment with BzATP didnot induce apoptosis. See, FIG. 2B. In contrast, baseline apoptosis ofN-Myc-hP2X₇-MDCK cells was 1.5-fold greater than of WT-MDCK cells andtreatment with BzATP augmented the apoptosis 2.5-fold. See, FIG. 2C;Wang et al, 2004a. Treatments of WT-MDCK cells with anti-sense P2X₇oligonucleotides or with random-control P2X₇ oligonucleotides had noeffects on apoptosis. See, FIG. 2B. In contrast, treatment ofN-Myc-hP2X₇-MDCK cells with anti-sense P2X₇ oligonucleotides, but notwith random-control P2X₇ oligonucleotides, inhibited both the baselineapoptosis and the BzATP-augmented apoptosis. See, FIG. 2C. These dataindicate that the P2X₇ receptor is both sufficient and necessary tomediate BzATP-dependent apoptosis.

3. In Vivo BzATP Effects on Normal Tissues

P2X₇ immunoreactivity in the mouse skin, that is morphologically andbiochemically similar to human skin, was found predominantly in thegerminative basal/parabasal epidermal cells and in the germinative basalcells of the hair shafts and hair roots. See, FIGS. 1F and 1G. Moreover,P2X₇ receptor expression levels in normal epidermal cells, culturedcells, and in in vivo mouse skin were similar to those reported in othertypes of normal human epithelia. See, FIGS. 1C and 1F; Li et al, 2006;and Li et al, 2007. Also, in human uterine epithelia the growth ofepithelial cells has been reported to be controlled by P2X₇-mediatedapoptosis. Wang et al, 2004a; Li et al, 2006; and Li et al, 2007.

a. In Vivo Tolerability and Short-Term Effects of BzATP

BzATP (100 μM in 200 μl [20 nmol, 14.3 μg, 1.0 μg/cm²]), was appliedtwice weekly for 4 weeks on the posterior region of the shaved dorsalskin. The concentration/dose of BzATP and frequency of treatments werechosen based on the above discussed cell culture data. Each animalserved as its own control by having vehicle-containing solution appliedtwice weekly for 4 weeks on the anterior region of the shaved dorsalskin.

Animals tolerated the treatments uneventfully, and there were nonoticeable changes in physical characteristics, animals' behavior,feeding habits or in animals' weights (not shown). Treatments with BzATPor the vehicle solution also had no visible effect on the dorsal skin.See, FIG. 13A. Histological H&E evaluation showed no differences incross sections obtained from the posterior (BzATP) or anterior (Control)skin dorsal areas. See, FIGS. 13B and 13C. In cross sections of theposterior (BzATP-treated) region of the dorsal skin, there was anincreased number of epidermal basal/parabasal P2X₇-positive cells withnuclei at different stages condensation, fragmentation and pyknosis.See, FIG. 3D, arrows. These data suggested that treatment with BzATPaugmented apoptosis of epidermal cells.

Also studied were the effects of local treatment with BzATP on skinapoptosis in vivo by TUNEL staining. The negative control experimentshowed minimal auto-fluorescence in cross sections of the mouse skin.See, FIGS. 13D and 13E. In skin cross sections of non-treated mice, onlyfaint TUNEL staining decorated the epidermis. See, FIG. 13F. Incontrast, in skin cross sections of BzATP-treated mice, numerousepidermal basal/parabasal cells and epidermal hair shaft cells stainedTUNEL positive. See, FIG. 13G. Additionally, DAPI stains of crosssections of BzATP-treated skin revealed greater proportion of nuclei atadvanced stages of condensation, fragmentation and pyknosis as comparedto controls. See, FIG. 13I and FIG. 13H, respectively.

b. In Vivo Long-Term Effects of BzATP

BzATP was applied locally twice a week on the surface of the shaveddorsal skin for a total of 16 weeks. The control group included micethat had their back shaved and were treated only with the vehicle.Long-term BzATP treatment had no noticeable effect on animals' grossappearance of the treated skin. See, FIGS. 14A & 14C versus FIGS. 14B &14D, respectively. Further, no effects were observed on behavior,feeding habits, or body weights (data not shown). Mean serum levels ofALT (SGPT) and AST (SGOT) were similar among the four groups, and werein the normal range for the mouse (not shown).

Histological evaluation of cross sections of dorsal skin showed nodifferences in specimens obtained from animals in the control and BzATPgroups. See, FIGS. 3G and 3H, and FIGS. 14E and 14F. Also, there were nosignificant differences in Ki67 immunoreactivity in cross sectionsobtained from the control and BzATP groups. See, FIGS. 3I and 3J. Incontrast, in the BzATP group TUNEL staining was significantly enhancedin epidermal and in hair shaft keratinocytes as compared to the controlgroup, and TUNEL staining co-localized with P2X₇ immunoreactivity. See,FIGS. 3K-3X.

In the epidermis of BzATP-treated mice, the TUNEL staining was foundmainly in basal/parabasal layers. See, FIGS. 3L and 3R, horizontalarrows; FIGS. 3V and 3X, and FIGS. 14G and 14H. Furthermore, enhancedTUNEL staining co-localization with P2X₇ immunoreactivity was primarilyconfined to the basal/parabasal layers. See, FIGS. 3T-3X, FIGS. 14I and14J (low magnification) and FIGS. 14K and 14L. (high magnification).Image analysis of TUNEL staining in the epidermis in cross sections fromthe BzATP group revealed mean pixel density (±SD) of 5.2±0.3 versus0.8±0.2 in epidermal cross sections from the control group (p<0.01,n=3).

In the skin of BzATP-treated mice, TUNEL staining was also significantlyenhanced in hair shaft keratinocytes compared to the control group. See,FIGS. 3L and 3R, vertical arrows; and FIGS. 3V and 3X. TUNEL stainingalso was co-localized with P2X₇ immunoreactivity. See, FIGS. 3T-3X.Image analysis of TUNEL pixel density in hair-shaft cross sections fromthe BzATP group was 6.1±0.8 versus 1.1±0.3 in hair-shaft cross sectionsfrom the control group (p<0.01, n=3).

Collectively, these data indicate that treatment with BzATP up-regulatedapoptosis in proliferating epidermal and hair shaft keratinocytes.However, in the normal mouse skin, the BzATP treatment and the augmentedapoptosis did not affect morphology or histology of the skin.

4. In Vivo BzATP on Cancerous Tissue Development

Experiments were performed to test the hypothesis that activation of theP2X₇ receptor could inhibit development of epidermal neoplasia. Theseexperiments utilized the mouse two-step DMBA/TPA skin neoplasia model,which involves tumor initiation by local treatment with DMBA, followedby tumor promotion by local treatment with TPA. Agarwal et al, 2005; andGuo et al, 2006. Mice had their dorsal skin shaved, and DMBA was appliedonce by topical application onto the shaved dorsal skin. TPA treatmentby topical application onto the shaved dorsal skin was started one weeklater and continued twice a week for 12 weeks.

Epithelial cancers usually develop from a premalignant lesions, e.g., apapilloma, and the cancer risk of premalignant epithelial lesions mayvary from 0.1% to 20%. 48. Reibel J, “Prognosis of oral pre-malignantlesions: Significance of clinical, histopathological, and molecularbiological characteristics” Crit Rev Oral Biol Med 2003, 14:47-62; Fu etal., “The actinic (solar) keratosis: A 21st century perspective” ArchDermatol 2003, 139:66-70; and Lindeque B G, “Management of cervicalpremalignant lesions” Best Pract Res Clin Obstet Gynaecol 2005,19:545-561.

Animals were divided into three groups: Control mice (n=15) that hadtheir back shaved and were treated only with the vehicle;DMBA/TPA-treated mice (n=15); and DMBA/TPA-treated mice that wereco-treated with BzATP twice a week from week-2 (i.e. two weeks prior toDMBA) (n=14). All treatments were applied locally on the shaved dorsalskin. Papilloma development was monitored weekly from week 5 to 12 afterthe DMBA. Endpoints were percent animals with at least one papilloma;number of papilloma per animal; and mean papilloma size (mm [largestlesion dimension]). None of the animals in the control group haddeveloped papillomas, and they will not be further discussed.

Fourteen (14) out of the fifteen (15) animals in the DMBA/TPA group(93%) and twelve (12) out of the fourteen (14) (78%) animals in theDMBA/TPA+BzATP group developed at least one skin papilloma at week 12.See, FIG. 5A. Using time-to-event data (i.e., for example, aKaplan-Meier analysis for “papilloma-free” states) the log-rank testbetween the DMBA/TPA and DMBA/TPA+BzATP groups was not significant(p=0.273). However, analysis of the proportion having a papilloma atweeks 5-12 separately gave a borderline (p=0.055) result at week 6 oftreatment. Specifically there were 13/15 (86.7%) in the DMBA/TPA groupversus 7/13 (53.8%) in the DMBA/TPA+BzATP group having at least onepapilloma.

In the DMBA/TPA and DMBA/TPA+BzATP groups the mean number of papillomasper animal increased over the 12 week study period, but the increase inpapillomas in the DMBA/TPA+BzATP group was smaller than in the DMBA/TPAgroup. See, FIG. 5B. An independent samples t-test for weeks 5-12 forthe DMBA/TPA and DMBA/TPA+BzATP groups revealed borderline significantdifference at weeks 8 and 9 (p=0.051, 0.057) and a significantdifference at week 10 (2.3±0.34 and 1.23±0.34 papillomas per animal[mean±SEM], respectively, p=0.033). Repeated measures analysis ofvariance (ANOVA) yielded a significant time effect (p<0.01), aborderline group effect (p=0.067) and a non-significant time*groupinteraction effect (p=0.290), for the DMBA/TPA and DMBA/TPA+BzATPcurves. The latter indicates parallel non-interacting trends for theDMBA/TPA and DMBA/TPA+BzATP curves.

In the DMBA/TPA and DMBA/TPA+BzATP groups, the mean papilloma size peranimal increased over the 12 weeks study period, but the increase in theDMBA/TPA+BzATP group was smaller than in the DMBA/TPA group. See, FIG.5C. An independent samples t-test for weeks 5-12 for the DMBA/TPA andDMBA/TPA+BzATP groups revealed significant differences at all weeks forthe mean size (with respective p values ranging from 0.005 to 0.029).Thus, for example, at week 12 mean papilloma size (mm) per animal was5.86±0.91 versus 3.46 ±0.73 (mean±SEM), respectively (p=0.01). Likewise,repeated measures ANOVA yielded a significant time effect (p<0.01), asignificant group effect (p=0.011) and a non-significant time*groupinteraction effect (p=0.113), for the DMBA/TPA and DMBA/TPA+BzATPcurves. The latter indicates, again, parallel non-interacting trends forthe DMBA/TPA and DMBA/TPA+BzATP curves.

Interestingly, papillomas induced by DMBA/TPA treatment in miceco-treated with BzATP were less hypertrophic and displayed lessfrequently ulceration and necrosis. See, FIG. 5A(c). Also, in these micethe formed papillomas frequently showed various degrees of involution.See, FIG. 5A(c), arrows.

Collectively, these data show that in DMBA/TPA-treated mice,co-treatment with BzATP applied locally on the skin tended to decreasethe incidence of papilloma formation; it decreased the mean number ofpapillomas per animal by about 25% and the mean papilloma size by about45%. In addition, in mice co-treated with BzATP, formed papillomasunderwent more frequently involution.

Large papillomas (i.e., for example, greater than 5 mm in diameter) werebiopsied at week 10 from one animal of each of the two treatment groups.These tissues were assayed for microscopic H&E evaluation, Ki67immunostaining, and TUNEL. There were no differences among the twogroups in tissue architecture or histology or Ki67 immunoreactivity.See, FIG. 5B(a,b); and not shown, respectively. Papilloma tissues fromanimals in the DMBA/TPA group showed weak TUNEL staining. See, FIG.5B(c). In contrast, papilloma tissues from animals in the DMBA/TPA+BzATPgroup showed intense TUNEL staining in basal/parabasal regions of thepapilloma epithelial regions. See, FIG. 5B(d), arrow).

After twenty-eight (28) weeks, lesions induced by the localadministration of DMBA/TPA progressed into formation of squamousspindle-cell carcinomas. See, FIGS. 6 and 7. As the data presented belowshow, about one-third of the papillomas involuted after week 14 and theremaining persisted either as non-cancerous papillomas, or transformedto cancerous lesions. All cancerous lesions arose from pre-existingpapillomas, while none of the animals in the control group had developedskin lesions. See, FIG. 6A. There were no significant differences in themorphological or histological characteristics of the unaffected normalskin in the DMBA/TPA and the DMBA/TPA+BzATP groups. See, FIGS. 6A-I andFIGS. 7A-B, respectively. Similarly, there were no significantdifferences in the morphological and histological characteristics ofpapillomas in the DMBA/TPA and DMBA/TPA+BzATP groups. See, FIGS. 6B-Eand FIGS. 7C-D, respectively.

After week 14, some papillomas remained intact while other started toinvolute in both the DMBA/TPA and DMBA/TPA+BzATP groups. See, FIGS.6D-E, and FIG. 7E. However, in both groups most papillomas (i.e., forexample, about two-thirds) underwent cancerous transformation tosquamous cell carcinomas with spindle-cell changes. See, FIGS. 7F-I.There were no significant changes in the morphological and histologicalcharacteristics of cancers in the two groups. See, FIGS. 6F-I and notshown, respectively.

Overall, the data show that co-treatment with BzATP, applied locally onskin areas exposed to DMBA/TPA altered the incidence and pattern of skinlesions having progression to skin cancer. To evaluate the effects ofBzATP, changes in skin lesions in the DMBA/TPA and DMBA/TPA+BzATP groupswere compared relative to the length of treatment. Since formation ofpapillomas and cancerous lesions was time-related (i.e., for example, amarked cut-off occurs between weeks 13-14; see, FIG. 8A), data wereanalyzed separately for weeks 0-12 and weeks 14-28.

During weeks 0-12, the proportion of living animals with papillomastended to be lower in the DMBA/TPA+BzATP group than in the DMBA/TPAgroup, and analysis of the proportion of living animals having apapilloma separately was significant at week 5 of treatment ((p<0.05):48±12% versus 80±10%, respectively). The proportion of living animalswith any skin lesion between weeks 14-21 was similar in the two groups.Nonetheless, the proportion of living animals with any skin lesiondiffered significantly among the groups in weeks 22-28. See, FIG. 8A.

During the 28 week time period, the proportion of living animals withnon-cancerous lesions (i.e., for example, existing and involutingpapillomas) decreased in both groups. In contrast, the proportion ofliving animals with cancerous lesions in the DMBA/TPA group increasedsteadily, while in the DMBA/TPA+BzATP group the proportion of livinganimals with cancerous lesions decreased over time. For example, in week28 the proportions of living animals with cancerous lesions in theDMBA/TPA and the DMBA/TPA+BzATP groups were 100% and 43±9%,respectively. See, FIG. 8B.

In both groups, the mean number of papillomas per living animalincreased between weeks 0-12, but the increase in the DMBA/TPA+BzATPgroup tended to be smaller than in the DMBA/TPA group. See, FIG. 9A. Anindependent samples t-test revealed a significant difference at week 10(i.e., for example, 2.3±0.5 and 1.2±0.4 papillomas per animal [mean±SD],respectively, p<0.04). Also, a repeated measures analysis of varianceyielded a significant time effect for the DMBA/TPA and DMBA/TPA+BzATPcurves between weeks 0-12 (p<0.01). See, FIG. 9A. Between weeks 14-28,the mean number of total lesions per living animal was not significantlydifferent between the two groups. See, FIG. 9A. In both groups, the meannumber of non-cancerous lesions decreased over the 14-28 weeks period.See, FIG. 9B. The mean number of cancerous lesions, however, remainedthe same. See, FIG. 9C.

Animals in both groups were compared relative to the total size oflesions (in mm³) per animal. In both groups, the mean total papillomassize per living animal increased between weeks 0-12, but the increase inthe DMBA/TPA+BzATP group was smaller than in the DMBA/TPA group. See,FIGS. 6B, 6C, and 10A. An independent samples t-test revealedsignificant differences at all weeks for mean total papillomas size(p<0.01-0.03). See, FIG. 10A. For example, in week 12 mean totalpapillomas size (in mm³) per animal was 5.8±1.1 versus 3.4±1.0(mean±SD), respectively (p<0.01). Likewise, a repeated measures analysisof variance yielded a significant time effect (p<0.01); a significantgroup effect (p<0.02); and a non-significant time*group interactioneffect (p>0.1), for both DMBA/TPA and DMBA/TPA+BzATP data sets. Thenon-significant time*group interaction term, indicates non-interactingtrends between the DMBA/TPA and DMBA/TPA+BzATP groups.

Between weeks 14-28, the variability of the lesion sizes among the twogroups was large. Although it is not necessary to understand themechanism of an invention, it is believed that this variability was dueto an unproportional excessive growth of some lesions relative toothers. See, FIGS. 6D-6I. This precluded a comparison of the means oflesion size among the two groups. However, since most non-cancerouslesions in both groups tended to be smaller than 10 mm³ and theproportion of animals with non-cancerous lesions of >10 mm³ was low inboth groups (i.e., for example, <10%). See, FIG. 10B, triangles. Datarepresenting the proportion of living animals with cancerous lesions >10mm³ were compared among the two groups. After week 28, a significantlysmaller proportion of living animals was observed in the DMBA/TPA+BzATPgroup with cancerous lesions >10 mm³ than in the DMBA/TPA group. Forexample, in week 28 the proportion of living animals with cancerouslesions >10 mm³ were 81%±8% as compared to 16±4% in the DMBA/TPA and theDMBA/TPA+BzATP groups, respectively. See, FIG. 10B.

Five (5) mice in the DMBA/TPA+BzATP group survived despite havingdeveloped relatively large cancerous lesions, while maintaining normalweight and exhibiting normal behavior. See, FIG. 6I. In contrast, mostmice in the DMBA/TPA group with smaller cancerous lesions met IACUCeuthanization requirements due to poor general condition and excessivetumor burden. See, FIG. 6H. Analysis of the proportion of living animalswith cancerous lesions >200 mm³ showed a tendency for higher proportionof animals in the DMBA/TPA+BzATP group than in the DMBA/TPA group, butthe differences did not reach statistical significance. See, FIG. 10C.

Using time-to-event data analysis it was found that development ofcancerous lesions was significantly slower and lower in theDMBA/TPA+BzATP group than in the DMBA/TPA group. See, FIG. 11A. Survivalcurves for the DMBA/TPA and DMBA/TPA+BzATP groups were generated basedon event (i.e., for example, death from cancer) and time-to-event (inweeks) for each group. A log-rank test was used to compare the survivalcurves based on group. The overall survival rates among the two groupsdid not differ statistically, although there was a tendency for earlierdeath in the DMBA/TPA group as compared to the DMBA/TPA+BzATP group.See, FIG. 11B.

5. Mechanism of BzATP-Augmented Apoptosis

a. P2X₇ Receptor Expression

To better understand the mechanism of BzATP pro-apoptotic skin effects,experiments utilized cultured primary mouse keratinocytes that wereobtained from wild-type mice, and from the P2X₇-receptor-deficient P2X₇^(−/−)Pfizer (PO mice and P2X₇ ^(−/−)GSK mice.

In wild-type mouse keratinocytes, BzATP augmented apoptosis in adose-related manner. Effects began at BzATP levels as low as 50 nM,reaching maximal effect at 100-250 μM with an estimated BzATP EC₅₀ ofabout 10 μM. See, FIG. 16A. Pre-treatment with P2X₇-receptor anti-senseoligonucleotide decreased expression of the P2X₇-receptor. See, FIG.16B, insert. The P2X₇-receptor antisense oligonucleotide also inhibitedbaseline apoptosis and blocked the pro-apoptotic effect of BzATP. See,FIG. 16B. Although it is not necessary to understand the mechanism of aninvention, it is believed that the pro-apoptotic effect is likelyinduced paracrinologically by ATP secreted by the cells. Pre-treatmentwith random-control oligonucleotides had no effect on P2X₇-receptorexpression. See, FIG. 16B, insert. Baseline apoptosis or apoptosisinduced by BzATP was also unaffected by random-control oligonucleotides.See, FIG. 16B.

The dependence of the pro-apoptotic effect of BzATP on the expression ofthe P2X₇ receptor was further demonstrated in experiments usingkeratinocytes obtained from P2X₇-receptor-deficient mice. Compared towild-type mouse keratinocytes, in both the P2X₇ ^(−/−)Pf and P2X₇^(−/−)GSK keratinocytes, treatment with 100 μM BzATP failed to induceapoptosis. See, FIG. 16C.

b. Calcium Pore Formation

In one embodiment, the present invention contemplates a method fortreating cancer comprising activation of a P2X₇-mediated calcium pore(i.e., for example, a calcium channel). Although it is not necessary tounderstand the mechanism of an invention, it is believed thatP2X₇-dependent apoptosis may be mediated by calcium influx viaP2X₇-controlled pores, and involves the mitochondrial caspase-9 pathway.

In uterine epithelial cells, as well as in other types of cells,P2X₇-receptor-dependent apoptosis involves agonist-induced acute calciuminflux via P2X₇ pores. To understand whether BzATP-induced apoptosis inmouse keratinocytes involves formation of P2X₇ pores, experimentscompared activation by BzATP of the P2X₇ receptor by measuring cytosoliccalcium levels and the formation of P2X₇ pores by measuring ethidiumbromide influx. Feng et al., “A truncated P2X₇ receptor variant (P2X7-j)endogenously expressed in cervical cancer cells antagonizes thefull-length P2X₇ receptor through hetero-oligomerization” J Biol Chem2006, 281:17228-17237.

In mouse wild-type keratinocytes, treatment with 100 μM BzATP in a 1.2mM Ca²⁺ solution, induced an acute increase in cytosolic calcium, whichlasted at least 6 minutes. Conversely, when 1.2 mM EGTA was added to thesolution (i.e., a low calcium solution), BzATP induced a short termspike in cytosolic calcium that quickly returned to normal levels (i.e.,the prolonged, sustained, increase in cytosolic calcium was abolished).See, FIG. 17A. Although it is not necessary to understand the mechanismof an invention, it is believed that the short-term spike increase incytosolic calcium most likely represents calcium release fromintracellular stores. It is further believed that the lack of prolongedincrease in cytosolic calcium in cells bathed in a low calciumextracellular medium low indicates that the BzATP-induced prolongedincrease in cytosolic calcium involves calcium influx.

Experiments using mouse wild-type keratinocytes also revealed thattreatment with BzATP induced an acute increase in the influx of ethidiumbromide with a time-course similar to the increase in cytosolic calcium.See, FIG. 17B. Comparing the profiles of cytosolic calcium and ethidiumbromide demonstrated a similar dose-dependence for BzATP. See, FIG. 17C.Further, these profiles resembled the dose dependence of apoptosis onBzATP with threshold effects at 50-100 nM and pre-maximal responses at100-250 μM. See, FIG. 16A.

c. Cytosolic Calcium And Influx

Similar to the effects of BzATP on apoptosis, pre-treatment with theP2X₇-receptor anti-sense oligonucleotide blocked the BzATP-inducedincrease in cytosolic calcium and the BzATP-induced increase in ethidiumbromide. See, FIGS. 18A and 18B, respectively. Pre-treatment with therandom-control oligonucleotides had no effect on the responses to BzATP.

d. Extracellular Calcium Dependence

In mouse wild-type keratinocytes, lowering extracellular calciumattenuated baseline apoptosis and blocked the BzATP-induced apoptosis ina dose-related manner. See, FIG. 19A. Although it is not necessary tounderstand the mechanism of an invention, it is believed that theBzATP-augmented apoptosis involves caspase-9 and caspase-3. For example,treatment of mouse wild-type keratinocytes with a caspase-9 inhibitor(i.e., for example, LEHD-FMK) blocked BzATP-induced apoptosis while acaspase-8 inhibitor (i.e., for example, IETD-FMK) did not have asignificant effect. See, FIG. 19B. Positive controls including, but notlimited to, a specific inhibitor of terminal caspase-3 (i.e., forexample, DEVD-FMK) or a non-specific pan-caspase inhibitor (i.e., forexample, zVAD-FMK) similarly blocked BzATP-induced apoptosis. See, FIG.19B.

e. Effects on Cellular Proliferation

To determine if the development of the large cancerous lesions in someanimals in the DMBA/TPA+BzATP group was the result of a pro-mitogeniceffect of BzATP, rates of DNA synthesis (in terms of [³H]-thymidineincorporation) in response to BzATP were measured in mouse wild-typenormal keratinocytes. Pre-treatments with the P2X₇-receptor anti-senseP2X₇ oligonucleotides or the random-control oligonucleotides, andtreatments with BzATP had no significant effect on [³]-thymidineincorporation. See, FIG. 20.

The data showed that the main targets of BzATP in the normal skin areproliferating keratinocytes of the epidermal basal/parabasal layers andhair shafts. In these P2X₇-receptor expressing cells, BzATP augmentedapoptosis without evoking inflammatory changes. Experiments inP2X₇-deficient normal keratinocytes and in normal keratinocytes treatedwith antisense P2X₇ oligonucleotides showed that the P2X₇ receptor is anecessary mediator of the pro-apoptotic effect of BzATP, suggesting thatthe effect of BzATP is mediated by augmentation of P2X₇-mediatedapoptosis. Similar to normal skin, the main targets of BzATP inpapilloma tissues were P2X₇-receptor expressing proliferatingkeratinocytes at the base of developing papillomas. This finding relatesto the fact that in the mouse DMBA/TPA model, papillomas at risk fordeveloping into cancer are characterized by rapidly proliferatingkeratinocytes in the basal and parabasal layers of the papilloma. Glicket al., “The high-risk benign tumor: evidence from the two stage skincancer model and relevance for human cancer” Mol Carcinogenesis 2007,46:605-610.

Since treatment with BzATP decreased the incidence of DMBA/TPA-inducedpapillomas and their transformation into cancer, it is likely that thecellular mechanism of BzATP action involved augmented apoptosis ofproliferating papilloma keratinocytes bearing the potential of malignanttransformation. One of the differences between BzATP effects in thenormal skin and in papilloma tissues was the lack of macroscopic effectsin the former, while inhibiting the development and growth ofpapillomas. Thus, treatment with BzATP for 16 weeks in normal miceincreased apoptosis in proliferating keratinocytes but it did notproduce a thinning or ulceration of the skin, as would be expected of apotent pro-apoptotic drug.

Similarly, there were no significant differences in the morphologicaland histological characteristics of unaffected normal skin betweenanimals in the DMBA/TPA+BzATP group (BzATP treatment for 30 weeks) andthe DMBA/TPA group. However, in the DMBA/TPA+BzATP group, enhancedapoptosis was associated with inhibition of papilloma development. Thedisparity between BzATP effects between normal and papilloma tissuescould be related to differences in the growth rate of the respectivekeratinocytes. Normal skin cells are slow growing and their overallgrowth rate is apparently not affected by BzATP. In contrast, in thefast growing papilloma keratinocytes BzATP-induced apoptosis slows andinhibits growth. The data in normal mice also showed that localtreatment with BzATP had no adverse systemic effects, suggesting arelatively safe profile for the drug when applied locally on the skin.These data indicate that BzATP is absorbed from the skin into thebasal/parabasal epidermal regions and hair shafts. The data also suggestthat the predominant effect of BzATP is induction of apoptosis at thesite of application, targeting rapidly growing proliferatingkeratinocytes.

In contrast to papillomas, the expression level of P2X₇ receptors inDMBA/TPA-induced cancer cells was low, as was evident by three assays:in-situ immunoreactivity, Western blots, and qPCR. These findings aresimilar to those reported in non-melanoma skin cancer cells, uterine,bladder and breast epithelial cancers. Greig et al., “Expression ofpurinergic receptors in non-melanoma skin cancers and their functionalroles in A431 cells” J Invest Dermatol 2003, 121:315-327; Li et al.,“The P2X₇ Receptor: A novel biomarker of uterine epithelial cancers”Cancer Epidemiol Biomarkers Preven 2006, 15:1-8; Li et al., “Decreasedexpression of P2X₇ in endometrial epithelial pre-cancerous and cancercells” Gynecol Oncology 2007, 106:233-243; Zhou et al., “Micro-RNAsmiR-186 and miR-150 downregulate expression of the pro-apoptoticpurinergic P2X₇ receptor by activation of instability sites at the3′-untranslated region of the gene that decrease steady-state levels ofthe transcript” J Biol Chem 2008, 283:28274-28286; and Li et al., “P2X₇receptor expression is decreased in epithelial cancer cells ofectodermal, urogenital sinus, and distal paramesonephric-duct origin”(submitted, 2009). These findings suggest that the rapid proliferationof cancer cells could be in part due to the low expression of the P2X₇receptor and to attenuated P2X₇-mediated apoptosis. Treatment with BzATPaugmented apoptosis even in cancer cells expressing low levels of theP2X₇ receptor, but the effect was smaller than in normal or papillomacells.

Until recently, little was known about the relationship betweenP2X₇-receptor and cellular apoptosis. The data presented herein suggestthat P2X₇-receptor induced apoptosis may depend on enhanced calciuminflux via P2X₇ pores, and possibly mediated by thecaspase-9-mitochondrial pathway.

The following experimental findings in the present study support thishypothesis:

-   -   (a) Treatment with BzATP induced formation of pores and enhanced        calcium influx;    -   (b) the BzATP-induced apoptosis, pore formation and the        augmented and prolonged calcium influx were critically dependent        on the expression of the P2X₇ receptor;    -   (c) the BzATP-induced apoptosis, pore formation and the        augmented calcium influx had similar dose-dependence on BzATP;    -   (d) the BzATP-induced pore formation and the augmented calcium        influx began shortly (30-60 seconds) after adding BzATP. In        contrast, the BzATP-induced apoptosis required hours of        treatment with BzATP, commensurate with a gene-mediated effect;    -   (e) the BzATP-induced apoptosis depended on the presence of        extracellular calcium at a physiological concentration of 1.2        mM, and on calcium influx; and    -   (f) the BzATP-induced apoptosis could be blocked by co-treatment        with inhibitors of caspase-9 and caspase-3, but not of        caspase-8.        Since caspase-3 is a terminal step in the caspase cascade, a        possible interpretation of the present results is that        P2X₇-receptor-apoptosis is mediated by the caspase-9        (mitochondrial) pathway. Fawthrop et al.,” Mechanisms of cell        death” Arch Toxicol 1991, 65:437-444; and Soti et al.,        “Apoptosis, necrosis and cellular senescence: chaperone        occupancy as a potential switch” Aging Cell 2003, 2:39-45.        Collectively, the data presented herein suggest that        BzATP-dependent activation of the P2X₇ receptor involves        formation of pores in the plasma membrane, and that facilitated        uncontrolled influx of Ca²⁺ via the P2X₇ pores stimulates        apoptosis by the mitochondrial-caspase-9 pathway. P2X₇ pores are        believed to be formed of channels composed of pannexins and        ectodomains of the P2X₇ molecule, whose formation is dependent        upon a P2X₇ receptor/agonist interaction. Feng et al., “A        truncated P2X₇ receptor variant (P2X_(7-j)) endogenously        expressed in cervical cancer cells antagonizes the full-length        P2X₇ receptor through hetero-oligomerization” J Biol Chem 2006,        281:17228-17237; Locovei et al., “Pannexin1 is part of the pore        forming unit of the P2X₇R death complex” FEBS Lett 2007,        581:483-488; and Iglesias et al., “P2X₇ receptor-Pannexin1        complex: Pharmacology and signaling” Am J Physiol Cell 2008,        295:C752-C760.

Papilloma keratinocytes are shown herein to express the P2X₇ receptor;therefore, the high expression levels of the P2X₇ receptor in in vivopapilloma cells, and the significant apoptotic effects in response toBzATP, could explain the inhibitory effect of BzATP on papillomadevelopment. In contrast, the lesser effect of BzATP in skin cancercells could be explained by the low expression level of the P2X₇receptor in the cancer cells. At present, little is known whetherneoplastic transformation decreases P2X₇-receptor expression, or whetherneoplastic transformation is triggered preferentially in cells alreadyexpressing low levels of P2X₇ receptor. The former possibility issupported by data in endometrial and bladder cells where low expressionof the P2X₇ receptor was found already in pre-cancerous and earlycancerous cells but not in hyperplastic benign cells. Li et al.,“Decreased expression of P2X7 in endometrial epithelial pre-cancerousand cancer cells” Gynecol Oncology 2007, 106:233-243. Accordingly, thecarcinogenic process could have reduced P2X₇ expression during earlystages of cancer development. On the other hand, the possibility thatthe neoplastic transformation is triggered preferentially in cellsexpressing low levels of the receptor is supported by data in uterinecervical epithelia, wherein low expression of the P2X₇ receptor wasfound already in dysplastic cells. Li et al., “The P2X₇ Receptor: Anovel biomarker of uterine epithelial cancers” Cancer EpidemiolBiomarkers Preven 2006 15:1-8. Since only a small fraction of cervicaldysplasia cases progresses to cancer, it is possible that low expressionof the P2X₇ receptor in the cervix precedes the neoplastictransformation. Walboomers et al., “Human papillomavirus is a necessarycause of invasive cervical cancer worldwide” J Pathol 1999, 189:12-19:Raab et al., “Long-term outcome and relative risk in women with atypicalsquamous cells of undetermined significance” Am J Clin Pathol 1999,112:57-62; and Song et al., “Risk factors for the progression orpersistence of untreated mild dysplasia of the uterine cervix” Int JGynecol Cancer 2006, 16:1608-1613. Accordingly, abrogation ofP2X₇-mediated apoptosis could be responsible for the preservation ofgenetically aberrant cells that are susceptible to carcinogenic stimuli,favoring neoplastic transformation. Staibano et al., “Prognostic valueof apoptotic index in cutaneous basal cell carcinomas of head and neck”Oral Oncol 1999, 35:541-547.

The present data showed only partial inhibition (i.e., for example,˜50%) of papilloma and cancer formation in BzATP-treated mice. Arelatively low dose (i.e., 1 μg/cm² BzATP) was used in the in vitroexperiments. Therefore, it would be expected that higher doses, and/ormore frequent applications, could produce greater inhibition papillomasand cancers, both in size and frequency.

IV. P2X₇ Receptor Expression Regulation

In one embodiment, the present invention contemplates a method forupregulating P2X₇ receptor expression. Although it is not necessary tounderstand the mechanism of an invention, it is believed that suchreceptor expression upregulation increases the cell surface density ofP2X₇ receptors.

In one embodiment, the present invention contemplates a method fortreating and/or preventing cancer by increasing P2X₇ receptor cellsurface density. In one embodiment, the cell surface comprises a cancercell surface. In one embodiment, the cell surface comprises a papillomacell surface. In one embodiment, the increased P2X₇ receptor celldensity induces apoptosis.

A. Elucidation of the Active Promoter Region

To define the active promoter region and the Transcription InitiationStart Site (TpIS), a series of cDNA fragments were generatedencompassing a 1.7 kb DNA segment at the 5′ region of the human P2X₇gene. Nucleotides were numbered relative to the subsequently elucidatedTpIS (+1), which corresponds with nt 1683 of the human P2X₇ gene(GenBank Y12851). Initial experiments included cDNA fragments rangingfrom −1664/+32 to −53/+32 nt. See, FIG. 28 and FIG. 29A. The cDNAfragments were inserted into a luciferase vector, and theP2X₇-luciferase reporter was transfected into HEK293 cells which lackendogenous expression of the P2X₇. Significant promoter activity wasfound in fragments ranging from −1664/+32 to −158/+32 nt, while shorterfragments lacked significant promoter activity. A cDNA fragment of−1179/−380 nt lacked significant promoter activity, suggesting thatthere is little promoter activity upstream of nt −380. Since maximalpromoter activity was found in experiments using the −158/+32 ntfragment, the data suggested location of the active promoter of thehuman P2X7 gene in the −158/+32 nt region. See, FIG. 29A.

The TpIS was elucidated using the modified 5′ RACE method. Bysequencing, two possible TpISs were found upstream of the subsequentlydefined TpIS: Adenine bases at +1 nt and −73 nt. See, FIG. 28. Todifferentiate between the two, the TpIS-corresponding regions weremutated as (−1) CATT to GTAA, and (−73) AGGG to TATA. Fragments carryingthe mutated regions were inserted into the luciferase vector andtransfected into HEK293 cells. The results showed 70% luciferaseactivity in the (−73)AGGG→TATA construct but only 30% luciferaseactivity in the (−1)CATT→GTAA construct. See, FIG. 29B.

Sequence analysis also suggested two TATA-like sequences locatedupstream of the subsequently defined TpIS: TTAAA at −32 nt, and TTATC at−102 nt. See, FIG. 28. Mutation analysis revealed 50% luciferaseactivity in the (−32)TTAAA→TCCAA construct and no change in luciferaseactivity in the (−102)TTATC→CCATC construct. See, FIG. 29B. Collectivelythese data suggest a TpIS at site +1 nt and a TATA-like sequence TTAAAat site −32 nt.

B. Regions Distal to the TpIS Inhibit Transcription

Sequence analysis of the human P2X7 gene (GenBank Y12851) downstream ofthe active promoter revealed an unusually high concentration ofcytosine-phosphodiester-guanosine (CpG) dinucleotides sites in the +26to +573 nt region. This region of 547 nt contains 20 CpG sites, incontrast to the −158/+32 190 by active promoter region, which containsonly 4 CpG sites. Changes in methylation of cytosines within CpG siteshave been reported to modulate gene function. Chen et al., (2006)“Establishment and maintenance of DNA methylation patterns in mammals”Curr Top Microbiol Immunol 301:179-201. Consequently, changes in themethylation status of CpG sites in the region downstream of the activepromoter might regulate P2X₇ transcription. See, FIG. 28.

A construct containing the fragment −158/+573 resulted in lesstranscription compared to the −158/+32 construct as measured usingFluc/Rluc. See, FIG. 29C (upper panel). The data was replicated bymeasuring Fluc/GPDH mRNA as an endpoint. See, FIG. 29C (lower panel). Itcan be seen that data between the two different methods are similar,thereby indicating that cis regulatory elements contained within the +33to +573 nt region downstream of the active P2X₇ promoter may inhibittranscription.

C. Demethylation Increases P2X₇ Receptor Expression And Apoptosis

1. Aza-dC Increases P2X₇ mRNA Levels

To determine whether changes in DNA methylation modulate P2X₇ genetranscription, cultured cervical cells were treated with thede-methylation agent 5-aza-2′-deoxycytidine (Aza-dC), and effects onP2X₇ mRNA steady state levels were measured. For experiments, cells weretreated with 1 μM Aza-dC, which in preliminary experiments exerted nearmaximal effects (not shown).

Baseline levels of P2X₇ mRNA steady state levels (relative to CK-18)were higher in the normal hEVEC than in the HeLa cancer cervicalepithelial cells wherein treatment with Aza-dC increased P2X₇ mRNA bothin hEVEC and in HeLa cells. See, FIG. 30A. The effect wastime-dependent, and increases in P2X₇ mRNA were observed already 18-24hours after treatment. In hEVEC cells, levels of P2X₇ mRNA continued toincrease, while in HeLa cells P2X₇ mRNA levels leveled off after 24 hrsand began to decrease afterwards, but remained elevated as compared tobaseline for at least 72 hours after the start of treatment.

2. Aza-dC Increases P2X₇ Protein Levels

Aza-dC effects on P2X₇ protein were determined in terms of changes incellular immunoreactivity to the anti P2X₇ antibody. In non-treatedhEVEC cells, staining with the anti P2X₇ receptor antibody revealedhomogenous P2X₇ immunoreactivity. See FIG. 30B, insert. Furthermore,treatment with 1 μM Aza-dC for 48 hours increased the P2X₇immunoreactivity significantly. A similar effect was seen in HeLa cells(data not shown). Image analysis of P2X₇ immunofluorescence density inAza-dC-treated cells revealed a 2 fold increase after 48 hours oftreatment in P2X7 immunoreactivity for both the hEVEC and HeLa cells.See, FIG. 30B.

3. Aza-dC Increases P2X₇-Mediated Apoptosis

Baseline apoptosis was 2 fold higher in hEVEC cells than in HeLa cells.Treatment with the P2X₇-receptor specific agonist BzATP increasedapoptosis 2.5 fold in hEVEC cells but it had no significant effect inHeLa cells. Treatment with Aza-dC increased mildly baseline apoptosis inhEVEC cells, and significantly (2 fold) baseline apoptosis in HeLacells. In hEVEC cells pretreated with Aza-dC, co-treatment with BzATPresulted in greater apoptosis than in cells treated only with BzATP, butthe effect was mild. See, FIG. 30C.

In contrast, in Aza-dC-pretreated HeLa cells co-treatment with BzATPresulted in 2.5 fold greater apoptosis than in cells treated only withBzATP, and the degree of apoptosis was nearly that observed in hEVECcells under similar conditions. See, FIG. 30C. Although it is notnecessary to understand the mechanism of an invention, it is believedthat Aza-dC sensitized normal and cancer cervical cells to thepro-apoptotic effect of BzATP, probably by upregulating the expressionof P2X₇ mRNA and protein receptor levels.

To determine whether apoptosis per se can stimulate an increase in P2X₇mRNA, hEVEC and HeLa cells were incubated in serum-free medium for 14hours. Wang et al., (2005) “EGF facilitates epinephrine inhibition ofP2X₇-receptor mediated pore formation and apoptosis: a novel signalingnetwork” Endocrinology 146:164-174. The results (not shown) indicatedthat P2X₇ mRNA steady state levels in hEVEC and in HeLa cells weresimilar in serum-deprived and in serum-exposed cells.

D. P2X₇ Gene Methylation Status

Effects of changes in the methylation status on P2X₇ transcription wereevaluated in HEK293 cells transfected with the luciferase P2X₇ −158/+32and/or −158/+573 reporter constructs. Hypermethylation assays involvedincubation of the test plasmids with the CpG-Methylase M.SssI prior totransfections, and de-methylation assays were done by treatingtransfected cells with Aza-dC.

Hypermethylation had no significant effects on luciferase mRNA levels inHEK293 cells transfected either with the luciferase P2X₇ −158/+32 orluciferase −158/+573 reporter constructs. See, FIG. 31. De-methylation,induced by treatment with Aza-dC also had no significant effect onluciferase mRNA levels in cells expressing the −158/+32 nt construct. Incontrast, Aza-dC increased luciferase mRNA levels two fold in cellsexpressing the −158/+573 nt construct. See, FIG. 31.

Methylation status may not be a non-specific promoter regulator. Forexample, measuring effects of de-methylation and hypermethylation onCK-18 and GAPDH mRNA steady-state levels following treatment with Aza-dChad no significant effects on the steady-state levels (data not shown).Also, in HEK293 cells transfected with either the luciferase P2X₇−158/+32 or the −158/+573 reporters, de-methylation and hypermethylationassays did not affect significantly steady-state levels of GAPDH mRNA(data not shown).

Collectively, these data suggest that regions downstream of the activeP2X₇ promoter may regulate transcription by modulating DNA methylation.

E. CpG Sites and P2X₇ Gene Methylation Status

1. Downstream CpG sites Inhibit P2X₇ Transcription

CpG sites in a 547 nt region downstream of an active P2X₇ promoter (nt−158/+32) were evaluated using cDNA fragments were inserted into aluciferase vector. For example, a P2X₇-luciferase reporter wastransfected into HEK293 cells, and P2X₇ transcription was determined interms of luciferase activity. See FIG. 32.

Luciferase activity in the fragment −158/+221 was low, about 10%compared to that of the active promoter alone (nt −158/+32). Luciferaseactivity in the fragment −158/+232 was higher, about 75% compared to theactive promoter. Luciferase activities in fragments −158/+337 and−158/+402 were lower by 25% and 50%, respectively than in fragment−158/+232. Luciferase activity in fragments −158/+470 and −158/+503 wassimilar to that in fragment −158/+32. Luciferase activity in fragment−158/+573 was low, about 10% of that in fragment −158/+32 (the activepromoter). See, FIG. 32A.

To determine if CpG sites in the 547 nt region downstream of thepromoter affect P2X₇ transcription, selected CpG sites were mutated.See, FIG. 32B. The following mutations resulted in a significantincrease in luciferase activity: +211/+212 (CG/AA), +257/+258 (CG/TT),+278/+279 (CG/TT), +319/+320 (CG/AT), +330/+331 (CG/AT), +424/+425(CG/TT), +453/+454 (CG/TT), and +461/+464 (CGCG/ATTA). These datasuggest that CpGs +211/+212, +257/+258, +278/+279, +319/+320, +330/+331,+424/+425, +453/+454, and a bi-CpG complex +461/+464 inhibit P2X₇transcription.

2. Constitutive Hypermethylation of Downstream P2X₇ CpG Sites

A CpG-rich 547 nt region downstream of an active P2X₇ promoter maymodulate transcription based upon changes in methylation status.Methylation status of cytosines within selected CpG sites in the 547 ntregion was measured in: i) normal cultured human uterine cervicalepithelial cells; ii) cancerous cultured human uterine cervicalepithelial cells; iii) normal biopsied human cervix epithelial tissues;and iv) cancerous biopsied human cervix epithelial tissues.

cDNA segments of interest (i.e., for example, Segments 1-3) weregenerated from Aza-dC-treated HeLa cells and analyzed by the genomic DNAbisulfite conversion method followed by gene specific PCR andrestriction enzyme cutting. The amplified segments were cut withrestriction enzymes to detect potential methylation-sensitive cytosinesat CpG sites. Sites +193/+194 and or +211/+212 (Segment-1), and+330/+331 (Segment-2) were cut with MaeII; sites +461/+462 and or+463/+464 (Segment-3) were cut with BstUI. See, FIG. 28.

Demethylation with Aza-dC decreased restriction enzyme cleavage at CpGsites in all three segments. Densitometry scans showed that the degreeof cleavage at CpG sites (defined in terms of the ratio of densitometryof the cleaved band, relative to the uncleaved plus cleaved bands [%])decreased in Segment-1 from 22% to 5%; in Segment-2 from 65% to 43%; andin Segment-3 from 48% to 22%. These data suggest that Aza-dC inducedde-methylation of cytosines within CpG sites at the 547 by region, andconfirmed the validity of the method that was used. See, FIG. 33A.

Human placental genomic DNA treated in vitro with the CpG methylase SssIwas used as a positive control. See, FIG. 33B. Aliquots of placental DNAwere mixed with different amounts of SssI and the degree of methylationwas determined in terms of cleavage at CpG sites. The degree ofmethylation in the absence of methylase SssI was small, but it increasedin a dose-related manner relative to the amount of Sss1 versus placentalDNA in the reaction mixture. Compare, FIG. 33A and FIG. 33B.

Methylation status of cytosines within those CpG sites was comparedbetween in vitro cultured normal and cancer human cervical cells and invivo normal and cancer human cervix epithelial tissues. See, FIGS.33C-E, and FIGS. 33F-I, respectively. The results revealed a greaterdegree of cleavage in cultured cancer cells than in cultured normalepithelial cervical cells. Similar experiments were done on human cervixepithelial tissues using specimens obtained by microdissection fromuterine tissue cross-sections. Experiments utilized paired specimensfrom ten patients, including in each case normal and squamous cellcarcinoma tissues. Sufficient amounts of tissues were available in ninecases for Segments 1 and 3 and in eight cases for Segment 2. Theseresults also showed a greater degree of cleavage in cancer tissues thanin normal tissues. See, FIGS. 33F-H.

Semi-quantitative analysis of the data in cultured cervical cells showeda 5-10 fold higher degree of cleaved fractions in all three segments incancer cells than in normal cells. See, Table 6.

TABLE 6 The degree of the cleaved fractions (in terms of the ratio ofdensitometry of the cleaved versus the uncleaved plus cleaved bands [%])at CpG sites +193/+194 (and/or +211/+212) (Segment 1); +330/+331(Segment 2); and +461/+462 (and/or +463 +464) (Segment-3) downstream theP2X7 active promoter in cultured cancer and normal cervical cells (means± SD of 3-4 experiments). Segment-1 Segment-2 Segment-3 Cancer CellsHeLa 24 ± 63 23 ± 4  23 ± 2 CaSki 55 ± 23 38 ± 13 20 ± 4 HT3 53 ± 17 35± 10 22 ± 4 SiHa 7 ± 3 12 ± 5  25 ± 5 Normal Cells (hEVEC)  2 ± 1*  1 ±2*  13 ± 3* *P < 0.01.These data suggest a greater degree of hypermethylation of cytosines atthe tested CpG sites in the cancer cervical cells than in the normalcervical cells. Similar trends were obtained in cervix epithelialtissues in vivo. Densitometry ratios of [cleaved]/[uncleaved pluscleaved] fractions in normal and cancer tissues were obtained from thesame patient. The results showed greater degree of cleaved fractions incancer cases than in the corresponding normal tissues in 8/9 cases ofSegment-1 (p<0.05); in 7/8 cases of segment-2 (p<0.01), and in 8/9 casesof Segment-3 (p<0.05). See, FIG. 33I.

Collectively, these data suggest that in cultured cervical cells and inthe cervix in vivo cytosines within CpG sites +193/+194 (and or+211/+212), +330/+331, and +461/+462 (and or +463/+464) in the 547 byregion downstream of the active P2X₇ promoter are hypermethylated to agreater degree in cancer cells than in normal epithelial cells.

F. Enhancer Region DNA-Protein Binding Sites

The data presented herein demonstrate that P2X7 promoter enhancerregions may bind transcription modulating factors. Further, CpG sitehypermethylation status mediation of transcription activity may beexplained by enhancer region proximity. Putative P2X₇ enhancer regionsincluding, but not limited to, nt +222/+232 and +401/+573 in the 547 byregion downstream of the active P2X7 promoter were shown to containprotein binding sites by using electrophoretic mobility shift assays(EMSA) using four amplified fragments, as described herein. See, Table 4(infra). EMSA identified a first shifted band in the +217/+237 fragment;a second shifted band in the +401/+530 fragment; and a third, fourth,fifth and sixth shifted bands in the +401/+573 fragment. See, FIG. 34.(FIG. 7). These data indicate the presence of DNA-protein complexes inthe +217/+237 and the +476/+573 regions.

G. P2X₇ Expression Regulator Proteins Bind to Methylated CpG Regions

In one embodiment, the present invention contemplates a method oftreating and/or preventing cancer by administering compounds capable ofbinding to a P2X₇ receptor gene enhancer region, wherein the enhancerregion increases P2X₇ receptor expression. In one embodiment, the P2X₇gene comprises an enhancer region (i.e., for example, +222/+232). In oneembodiment, the compounds modulate P2X₇ receptor expression regulatorproteins. In one embodiment, the regulator proteins may be selected fromthe group including, but not limited to, p300, Elk-1, E47, E11aE, E2F,or p53. In one embodiment, a sequence of the putative enhancer region+222/+232 comprises binding sites for regulator proteins selected fromthe group including, but not limited to, p300, Elk-1, E47, E11aE, E2F,or p53.

p300 has been reported to promote transcription by acetylating histonesand integrating signaling from enhancer and promoter regions. Li et al.,(2002) “Acetylation of p53 inhibits its ubiquitination by Mdm2” J BiolChem 277:50607-50611. Elk-1 has been reported to regulate transcriptionby phosphorylation in response to activation of mitogen-activatedprotein kinase (MAPK) pathways. Yang et al., (2006) “Convergence of theSUMO and MAPK pathways on the ETS-domain transcription factor Elk-1”Biochem Soc Symp 73:121-129. E47 has been reported to be a member of theE2 protein family encoded by the E2A gene and may regulate celldevelopment and differentiation. For example, a repression or absence ofE47 activity have been implicated in cancer development. Yang et al.,(2008) “E47 controls the developmental integrity and cell cyclequiescence of multipotential hematopoietic progenitors” J Immunol181:5885-5894. Bioinformatics analysis of a sequence of the putativeP2X₇ enhancer region +401/+573 revealed putative binding sites for p300,Elk-1 and E47, as well for EIIaE, E2F, and p53. EIIaE and E2F are alsomembers of the E protein family; both control cell cycle progression andover-expression of E2F-1 can activate apoptosis. Mathis et al., (1981)“Specific in vitro initiation of transcription on the adenovirus type 2early and late EII transcription units” Proc Natl Acad Sci USA78:7383-7387; Hamel et al., (1992) “Transcriptional repression of theE2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1gene” Mol Cell Biol 12:3431-3438; Zheng et al., (1999) “Structural basisof DNA recognition by the heterodimeric cell cycle transcription factorE2F-DP” Genes Dev 13:666-674; and Pardee et al., (2004) “Regulation in Sphase by E2F” Cell Cycle 3:1091-1094.

The p53 tumor suppressor transcription factor is believed to play a rolein the expression of genes involved in the regulation of cell cycleprogression and cell death. Laptenko et al., (2006) “Transcriptionalregulation by p53: one protein, many possibilities” Cell Death andDifferentiation 13:951-961. Further, there may be an association betweenactivation of the P2X₇ receptor and the p53 apoptotic pathway and/orbetween increased expression of the P2X₇ receptor and p53 proteinlevels. Schulze-Lohoff et al., (1998) “Extracellular ATP causesapoptosis and necrosis of cultured mesangial cells via P2Z/P2X₇receptors” Am J Physiol 275:F962-F971; and Turner et al., (2007)“Increased expression of the pro-apoptotic ATP-sensitive P2X7 receptorin experimental and human glomerulonephritis” Nephrol Dial Transplant22:386-395.

The data presented herein show that the putative enhancer +222/+232 and+401/+573 regions co-localize with, or are flanked by, constitutivelymethylated CpGs. Although it is not necessary to understand themechanism of an invention, it is believed that the association ofinhibitory CpGs with binding sites of putative enhancers suggests a geneexpression regulatory mechanism, such that hypermethylated CpGs inhibitP2X₇ transcription by modulating the interaction of enhancertranscription factors with their cognate DNA binding domains.Consequently, it is further believed that demethylation of these CpGswould increase P2X₇ transcription.

In one embodiment, the present invention contemplates a method fortreating and preventing cancer by modulating P2X₇ transcriptionregulation. See, FIG. 27. Transcription of the P2X₇ receptor may beregulated by cis-enhancer elements located in nt +222/+232 and +401/+573downstream of an active P2X₇ promoter, which contain binding sites fortranscription factors. Transcription of the P2X₇ receptor is negativelycontrolled by methylated CpG sites +193/+194 (and/or +211/+212),+330/+331, and +461/+462 (and/or +463+464) that flank the enhancers.Since transcription in cells expressing the

−158/+573 nt construct was lower compared to that in cells expressing anactive promoter (nt −158/+32), the data suggest that, under baselineconditions, the effect of the putative inhibitors surpasses that of theputative enhancer(s).

The present data demonstrate that cells may have devised mechanisms toregulate expression and activity of the P2X₇ receptor to avoid unneededapoptosis. For example, enhancers of transcription downstream of an P2X₇promoter may be controlled by neighboring hypermethylated CpG sites.Further, it has been reported that P2X₇ transcripts can be degradedpost-transcriptionally by the actions of micro-RNAs. Zhou et al., (2008)“Micro-RNAs miR-186 and miR-150 downregulate expression of thepro-apoptotic purinergic P2X₇ receptor by activation of instabilitysites at the 3′-untranslated region of the gene that decreasesteady-state levels of the transcript” J Biol Chem 283:28274-28286.Although it is not necessary to understand the mechanism of aninvention, it is believed that hypotheses explaining reduced expressionof the P2X₇ receptor, by hypermethylation of the CpGs in cancer cellsmay: i) be secondary to the cancer process; or ii) precede thecarcinogenic stimuli such that the abrogated apoptosis predisposes cellsto the development of cancer.

In summary, the data presented herein suggest that P2X₇ transcriptionmay be regulated by two groups of direction-dependent cis regulatoryenhancers located within a 547 nt region downstream of the activepromoter (i.e, for example, segments +222/+232 and +403/+573). In oneembodiment, the regulatory enhancers comprise co-localized or clusteredCpG sites. In one embodiment, the CpG site comprises an inhibitory ciselements. Although it is not necessary to understand the mechanism of aninvention, it is believed that an inhibitory cis element may beregulated by the amount of cytosine hypermethylation. In one embodiment,cervical cell CpG sites within the P2X₇ gene include, but are notlimited to +211/+212+330/+331 and +461/+464. In one embodiment, thecervical cell CpG sites are hypermethylated to a greater degree incancer cells than in the normal cervical cells. Although it is notnecessary to understand the mechanism of an invention, it is believedthat such increased CpG hypermethylation results in decreased P2X₇receptor expression, thereby decreasing cell apoptosis.

In summary, the above data suggest that:

-   -   Reduced expression of the P2X₇ receptor may be associated with a        reduced rate of apoptosis, thereby predisposing cells to the        development of cancer.    -   P2X₇ transcription may be regulated by enhancer regions located        within a 547 nt region (+26/+573) downstream of an active        promoter.    -   P2X₇ transcription may be controlled by the hypermethylation        status of cytosines at CpG sites that cluster and/or co-localize        with an enhancers' sites.    -   Low expression of the P2X₇ receptor in epithelial cancer cells,        e.g. uterine cervical cancer cells may be determined, in part,        by an inhibition of transcription enhancers (i.e., for example,        those enhancers located within a 547 nt region downstream of a        promoter) increasing the hypermethylation status of flanking CpG        sites.        V. Administration of P2X₇ Receptor Agonists

P2X₇ receptor agonists can be administered to a subject by any meanssuitable for delivering these compounds to a subject. For example, P2X₇receptor agonists can be administered by methods suitable enteral orparenteral administration route. Suitable enteral administration routesfor the present methods include, but are not limited to, oral, rectal,or intranasal delivery. Suitable parenteral administration routesinclude, but are not limited to, intravascular administration (i.e., forexample, intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstallation into the vasculature); peri- and intra-tissue injection(i.e., for example, peri-tumoral and intra-tumoral injection,intra-retinal injection, or subretinal injection); subcutaneousinjection or deposition, including, but not limited to, subcutaneousinfusion (i.e., for example, by osmotic pumps); direct application tothe tissue of interest, for example by a catheter or other placementdevice (i.e., for example, a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Another administration route includes, but is not limitedto, injection and/or infusion directly into a tumor.

Liposomes are used to deliver P2X₇ receptor agonists to a subject.Liposomes can also increase the blood half-life of the gene products ornucleic acids. Liposomes suitable for use in the invention can be formedfrom standard vesicle-forming lipids, which generally include neutral ornegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of factors suchas the desired liposome size and half-life of the liposomes in the bloodstream. A variety of methods can be used for preparing liposomes. Szokaet al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); and U.S. Pat. Nos.4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosuresof which are herein incorporated by reference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands which bindto receptors prevalent in cancer cells, such as monoclonal antibodiesthat bind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In one embodiment, a liposome of the invention cancomprise both opsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES. U.S. Pat. No. 4,920,016, the entiredisclosure of which is herein incorporated by reference.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include, but are notlimited to, polyethylene glycol (PEG) or polypropylene glycol (PPG)derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone;linear, branched, or dendrimeric polyamidoamines; polyacrylic acids;polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylicor amino groups are chemically linked, as well as gangliosides, such asganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, orderivatives thereof, are also suitable. In addition, the opsonizationinhibiting polymer can be a block copolymer of PEG and either apolyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, orpolynucleotide. The opsonization inhibiting polymers can also be naturalpolysaccharides containing amino acids or carboxylic acids, e.g.,galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid,pectic acid, neuraminic acid, alginic acid, carrageenan; aminatedpolysaccharides or oligosaccharides (linear or branched); orcarboxylated polysaccharides or oligosaccharides, e.g., reacted withderivatives of carbonic acids with resultant linking of carboxylicgroups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to a liposome membrane.For example, an N-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example solid tumors, will efficiently accumulate theseliposomes. Gabizon, et al., Proc. Natl. Acad. Sci., USA, 18:6949-6953(1988). In addition, the reduced uptake by the RES lowers the toxicityof stealth liposomes by preventing significant accumulation of theliposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miRNA gene products or miRNA gene expression inhibition compounds(or nucleic acids comprising sequences encoding them) to tumor cells.

VI. P2X₇ Receptor Agonist Pharmaceutical Formulations

P2X₇ receptor agonists compounds are preferably formulated aspharmaceutical compositions, sometimes called “medicaments,” prior toadministering to a subject. Pharmaceutical compositions of the presentinvention are characterized as being at least sterile and pyrogen-free.As used herein, “pharmaceutical formulations” include, but are notlimited to, formulations for human and veterinary use. Methods forpreparing pharmaceutical compositions of the invention are described.In: Remington's Pharmaceutical Science, 17th ed., Mack PublishingCompany, Easton, Pa. (1985), the entire disclosure of which is hereinincorporated by reference.

Pharmaceutical formulations contemplated by the present inventioncomprise at least one P2X₇ receptor agonist (e.g., 0.1 to 90% byweight), or a physiologically acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. Pharmaceutical formulations of theinvention may also comprise at least one P2X₇ receptor agonist which maybe encapsulated by liposomes and/or a pharmaceutically-acceptablecarrier. In one embodiment, a pharmaceutical composition comprises anP2X₇ receptor agonists including, but not limited to, BzATP. Preferredpharmaceutically-acceptable carriers are water, buffered water, normalsaline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include, but are not limited to, stabilizers,antioxidants, osmolality adjusting agents, buffers, and pH adjustingagents. Suitable additives include, but are not limited to,physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be usedincluding, but not limited to, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one P2X₇ receptor agonist. Apharmaceutical composition for aerosol (inhalational) administration cancomprise 0.01-20% by weight, preferably 1%-10% by weight, of the atleast one P2X₇ receptor agonist encapsulated in a liposome as describedabove, and a propellant. A carrier can also be included as desired;e.g., lecithin for intranasal delivery.

EXPERIMENTAL

All chemicals, unless specified otherwise, were obtained from SigmaChemical (St. Louis, Mo.). Primary antibodies for the immunostaining andWestern blots were as follows: Rabbit polyclonal anti-P2X₇ receptorantibody, which recognizes the functional full length P2X₇ receptor wasfrom Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDHantibody was from BD Transduction Laboratories (Lexington, Ky.). Thesecondary antibody was goat anti-rabbit Alexa Fluoro antibody(Invitrogen). Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK; SEQID NO:1), Ile-Glu-Thr-Asp-O-methyl-fluoromethylketone (IETD-FMK: SEQ IDNO: 2), Benzyloxy-valine-alanine-aspartate-O-methyl-fluoromethylketone(zVAD-FMK: SEQ ID NO: 3), and Asp-Glu-Val-Asp-O-methylfluoromethylketone(DEVD-FMK: SEQ ID NO: 4) were from Calbiochem La Jolla, Calif.), andwere used at a concentration of 50 μM.

Example I Human Keratinocyte Cell Cultures and Transfections

Primary cultures of human normal keratinocytes were generated fromdiscarded foreskins. Tissues were obtained following non-ritualcircumcision of newborn males at the nursery of the Department ofObstetrics and Gynecology, University Hospital CASE Medical Centeraccording to approved IRB protocol 08-06-28. Human epidermal squamouscell carcinoma-9 (SCC-9) cells and Madin Darby canine kidney cells(MDCK, strain II) were obtained from the ATCC. MDCK cells lackendogenous expression of P2X₇ receptor, and MDCK cells expressingtetracycline-regulated repressor (Feng et al, 2006) were used forheterologous transfection expression experiments.

For inducible expression of P2X₇ receptors in MDCK cells, the human P2X₇gene (NM_(—)002562), including a Myc tag attached to the N-terminus wassubcloned into pcDNA4/TO vector (Invitrogen) with Hind III and Not Isites. Primers, the method of transfections, and the generation ofstable MDCK clones were described (Feng et al, 2006). Stable clones weremaintained in tetracycline-free medium and 100 μg/ml zeocin. Expressionof Myc-P2X₇ genes was induced by 100 ng/ml doxycycline, and expressionof Myc-P2X₇ protein in stable MDCK cells was confirmed by RT-PCR (Fenget al, 2006). Cell cultures were maintained as described (Wang et al,2005; Feng et al, 2006; Li et al, 2006; Li et al, 2007).

Example II Protein Assays

Immunostaining and Western blots were described (Wang et al, 2005; Fenget al, 2006; Li et al, 2006; Li et al, 2007). Assays using human-derivedcells (keratinocytes and SCC-9) and MDCK cells utilized the rabbitpolyclonal anti-P2X₇ receptor antibody (Alomone Laboratories, Jerusalem,Israel); mouse anti-c-Myc antibody (Santa Cruz Biotechnology, SantaCruz, Calif.); mouse anti-E-Cadherin antibody (Invitrogen,http://www.invitrogen.com); and the anti-tubulin antibody (From theDevelopmental Studies Hybridoma Bank, University of Iowa, Iowa City,Iowa). Assays using mouse tissues utilized the rabbit anti-P2X₇ receptorantibody, the rabbit polyclonal anti Ki67 antibody (Dako,http://www.dako.com), and goat anti-rabbit Alex Fluoro 546 secondaryantibody (Invitrogen).

Image-analysis of immunofluorescence data was described (Li et al,2007). Representative fields were captured and saved in Adobe Photoshop.Pictures were scanned using UN-SCAN-IT (Silk Scientific, Orem Utah) bychoosing 3-5 representative fields for each picture. Light intensity ineach field was digitized and average pixel density per field wasdetermined using the program software.

Confocal laser microscopy was described (Feng et al, 2005). Briefly,MDCK cells were plated on 35-mm glass-bottom culture dishes (MatTekCorporation, Ashland, Mass.). Cells were immunostained with theanti-P2X₇ receptor antibody, and planar and vertical images wereobtained in-situ.

Example III P2X₇ mRNA In-Situ Hybridization

Primers for the anti-sense and sense probes of the full length P2X₇ cDNAtemplate were described (Li et al, 2006). For assays, cultured cellswere plated on Poly-lysine-coated coverslips and grown in culture mediumto subconfluence. Staining was evaluated using Nikon Eclipse 80imicroscope (Melville, N.Y.).

Example IV Real-Time PCR

Total RNA was extracted by RNeasy mini kit (QIAGEN, Valencia, Calif.)and one-step Real-time qPCR was carried out as described (Li et al,2006; Li et al, 2007). Primers for the P2X₇, E-Cadherin andcytokeratin-18 (CK-18) (used for data normalization) and PCR conditionswere described (Li et al, 2006; Li et al, 2007). Relative quantification(RQ) was calculated in terms of C_(t) as described (Li et al, 2006; Liet al, 2007).

Example V ATP Assays

Cells were grown on plates and maintained in a volume of 300 μl ofRinger's Solution. Twenty minutes after stabilization 50 μl aliquots ofconditioned medium were collected and assayed by a chemiluminescentmethod linked to Firefly luciferase-luciferin as described (Wang et al,2004a). Bubbled Ringer's solution was used as blank to determinebackground ATP, and ATP in the medium was determined from a standardcurve of samples with known ATP concentrations. The lower limit ofdetection for ATP was 0.25 nM.

Example VI Apoptosis Assays

Apoptosis may be quantified in terms of percent solubilized DNA (Wang etal, 2004a), or by using the commercial cell-death detection ELISA kit(Roche Applied Science, Nutley, N.J.) (Feng et al, 2006).

TUNEL assays in tissues cross sections were performed using DeadEnd™Fluorometric TUNEL System (Promega, Madison, Wis.) according to themanufacturer's protocol. For TUNEL-P2X₇ co-staining, tissue crosssections were first assayed for TUNEL, followed by P2X₇ immunostaining.The modified combined method resulted in only negligible crossfluorescence interference.

Apoptosis of cultured mouse keratinocytes was quantified in terms ofpercent solubilized DNA, or by using the commercial cell-death detectionELISA kit (Roche Applied Science, Nutley, N.J.). Wang et al.,“P2X₇-receptor mediated apoptosis of human cervical epithelial cells” AmJ Physiol 2004, 287:C1349-C1358; and Feng et al., “A truncated P2X₇receptor variant (P2X_(7-j)) endogenously expressed in cervical cancercells antagonizes the full-length P2X₇ receptor throughhetero-oligomerization” J Biol Chem 2006, 281:17228-17237, respectively.

Example VII P2X₇-Antisense Oligonucleotides

P2X₇-specific antisense oligonucleotides (ASO) and random controloligonucleotides (RCO) were designed from the published sequence of thehuman P2X₇ gene (NM_(—)002562) using a previously described method. Zhuet al, 2006. The sequences of the 20-mer ASO that would hybridize to thecoding region of exon 7 (nt 724-741), and the RCO were as follows:ASO-TTT CAG ATG TGG CAA TTC AG (SEQ ID NO: 5); RCO-TAT CAC ATC TCG CAATAG AC (SEQ ID NO:6). In the RCO, the antisense sequence was randomlyreplaced with adenine and thymine residues so that the oligonucleotideshad the same length (20-mer) and GC content (33%) as the ASO. The RCOwas designed such that no cross-hybridization against the P2X₇ genewould occur. To assess the effects of the ASO and RCO on P2X₇ mRNAexpression, cultured cells were treated with or without ASO or RCO atconcentrations of 10 μM. qPCR assays were carried out after 2 days, andWestern blots and apoptosis determinations were made after 3 days.

Example VIII Animal Experiments

This example evaluates to what degree pharmacological activation ofP2X₇-mediated apoptosis could inhibit cancer development in vivo. [55]

The experimental system was the mouse skin cancer model, based on therationale that both in rodents and in humans the P2X₇ system plays animportant role in the control of growth of keratinocytes [45-49,55].Skin lesions were induced by the Two-Step procedure, which involvestumor initiation with 7,12-dimethyl-benz(a)anthracene (DMBA), and tumorpromotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). See, FIG.23A. DMBA and TPA were applied locally on the shaved dorsal skin ofmice. Control animals were treated either with DMBA/TPA or with BzATPalone. Data were evaluated after 28 weeks of treatment (for the DMBA/TPAand DMBA/TPA+BzATP groups) or after 4 and 16 weeks (for the BzATP groupalone).

Experiments using mice were done according to an approved CWRU IACUC IRBprotocol [2006-0141]. Experiments utilized 6-8 weeks old FVB female mice(obtained from Charles River, Wilmington, Mass.). The animal's dorsalskin (about 3×5 cm) was shaved using Oster animal clipper (MountainHome, Ark.) followed by weekly application of Nair® hair remover lotion.Treatments included the application of one or more of the followingdrugs, directly onto the shaved dorsal skin: DMBA (50 μg/200 μl acetone[950 μM, 190 nmol, 3.3 μg/cm²]); TPA (3 μg/200 μl acetone [20 μM, 4nmol, 0.2 μg/cm²]); BzATP (14.3 μg/200 μl of 2.3/1 vol/vol solution ofpropylene-glycol [PG]/ethanol [EtOH][100 μM, 20 nmol, 1.0 μg/cm²]); orthe BzATP vehicle only (200 μl of PG/EtOH). DMBA was applied once, whileTPA and BzATP were applied twice a week. Skin neoplasia in the mice wereinduced by the “Two-Step” method, which involved tumor initiation bylocal treatment with 7,12-dimethylbenz(a)anthracene (DMBA), followed bytumor promotion with local treatment of12-O-tetradecanoylphorbol-13-acetate (TPA). Guo et al., “Disruption ofEphA2 receptor tyrosine kinase leads to increased susceptibility tocarcinogenesis in mouse skin” Cancer Res 2006, 66:7050-7058. Theconcentration/dose of BzATP and frequency of treatments were chosenbased on data in cultured cells (supra).

The shaved dorsal skin was divided into two equal anterior and posteriorareas. Each animal was treated with BzATP applied locally twice a weekfor between 4-16 weeks on the anterior skin area; vehicle only wasapplied in parallel twice a week for 4 weeks on the posterior skin area.At the end of the experiment animals were euthanized by cervicaldislocation and dorsal skin strips were obtained from each animal fromthe anterior and posterior skin areas for microscopic H&E tissueevaluation and P2X₇ immunostaining.

From each animal blood samples were obtained from the retro-bulbarvenous complex for ALT (SGPT) and AST (SGOT) assays. In addition, dorsalskin strips were obtained from each animal for microscopic H&E tissueevaluation, Ki67 immunostaining, and in-situ detection of DNAfragmentation by the TUNEL method.

In some groups of animals, cancerous lesions were generated by using aDMBA/TPA treatment, both with and without the concurrent administrationof BzATP. Control, no treatment; DMBA plus TPA; and DMBA/TPA plus BzATP.Treatments with BzATP began two weeks prior to the DMBA/TPA treatments.Endpoints were determined after 7-16 weeks. Endpoints were percentanimals with at least one papilloma; number of papillomas per animal;and mean papilloma size (mm's determined at the largest lesiondimension). Some animals underwent biopsy of papillomas using minirotary ElliptiPunch blade (HUOT, Menomonee Falls, Wis.). The biopsytissues were assayed for microscopic H&E tissue evaluation, Ki67immunostaining, and TUNEL.

Biopsy of skin papilloma was done on one living animal using mini rotaryElliptiPunch blade (HUOT, Menomonee Falls, Wis.). Criteria foreuthanasia (by cervical dislocation) prior to week 28 were according toMontgomery guidelines, including animals with excessive tumor burden, orwith ulcerated lesions regardless of size that were bleeding, necrosed,or infected. Hetherington et al., “Mouse care and husbandry” In: MouseGenetics and Transgenics: A Practical Approach Edited by: Jackson I J,Abbott C M. Oxford University Press; 2000:1-25. The experiment wasterminated at week 28, when all remaining living animals wereeuthanized. After death, representative samples were obtained from allskin lesions as follows: i) DMBA/TPA group—4 papillomas and 26 cancersamples; and ii) DMBA/TPA-BzATP group—2 papillomas and 12 cancersamples. All animals underwent postmortem exam for the presence ofmetastases and non-epidermal tumors (none were found).

Most lesions were defined as papillomas based on the actual histologicaldiagnosis in one case, the typical morphological appearance, and theextensive experience gained by others using this model and methodology.Guo et al., “Disruption of EphA2 receptor tyrosine kinase leads toincreased susceptibility to carcinogenesis in mouse skin” Cancer Res2006, 66:7050-7058; and Glick et al., “The high-risk benign tumor:evidence from the two stage skin cancer model and relevance for humancancer” Mol Carcinogenesis 2007, 46:605-610. Lesions biopsied during14-28 weeks were defined either as cancers (as diagnosed in each case atthe time of death), or as non-cancerous lesions, including existing orinvoluting papillomas (diagnosed at the time of death) or lesions thatdisappeared prior to death.

The main results were as follows:

-   -   (a) Local administration of DMBA/TPA induced skin papillomas at        weeks 5-12, and squamous spindle-cell carcinomas afterwards.        See, FIG. 23B-I.    -   (b) At weeks 0-12 the incidence of papillomas was lower by about        50% in animals co-treated with BzATP.    -   (c) The development of cancerous lesions was significantly        slower and lesser in the DMBA/TPA+BzATP group than in the        DMBA/TPA group. At week 28 the incidence of cancerous lesions        was lower by about 50% in animals co-treated with BzATP. See,        FIG. 24A.    -   (d) The survival rates showed a tendency for earlier deaths in        the DMBA/TPA group than in the DMBA/TPA+BzATP group. See, FIG.        24B.    -   (e) In the normal skin, P2X₇ immunoreactivity localized        predominantly within proliferating keratinocytes at the        basal/parabasal layers, and in hair shafts. See, FIG. 25A and        FIG. 25B.    -   (f) In papillomas, P2X₇ immunoreactivity was similar to normal        tissues, and it localized predominantly within proliferating        keratinocytes at the base of the developing papillomas. See,        FIGS. 25C and 25D.    -   (g) In cancer cells, P2X₇ immunoreactivity and P2X₇ mRNA levels        were 4-5 fold lower than in normal epidermal or papilloma cells.        See, FIGS. 25E-H.    -   (h) Treatment with BzATP up-regulated apoptosis of P2X₇ receptor        expressing cells in:        -   i) normal skin (See, FIGS. 26A-N);        -   ii) DMBA/TPA-induced papilloma keratinocytes (See, FIGS.            26O-R), and        -   iii) DMBA/TPA-induced cancer cells (See, FIGS. 26S-V).    -   The effect in cancer cells was smaller than in normal or        papilloma keratinocytes. See, FIG. 26.    -   (i) Treatment with BzATP had no significant effect on the        morphology and histological characteristics of the skin. See,        FIGS. 6A and 6B.    -   (j) The local treatment with BzATP had no significant effect on        the behavior of the animals, feeding habits, body weight, and        liver functions.

In summary, local treatment with the P2X₇-receptor-specific ligand BzATPdecreased the development of DMBA/TPA-induced skin papillomas and skincancers in mice by 50%. BzATP was observed to augment apoptosis inproliferating keratinocytes. Local treatment with BzATP did not producelocal or systemic adverse effects.

Example IX ALT (SGPT) and AST (SGOT) Assays

Assays used Liquid ALT and Liquid AST reagent sets (Cat # A7526-150a andA7561-150, respectively) obtained from Pointe Scientific (Canton Mich.),and were performed according to the supplier instructions.

Example X Data Analysis

The proportion of living animals with papillomas or cancerous lesionswas calculated at each week throughout the study period and comparedbetween groups by chi-square or Fisher's exact test. Time-to-event datawere used to assess differences in the formation of cancerous lesionsand the Kaplan-Meier method (with log-rank test) was used to comparedifferences between groups.

Mean number of lesions per animal was calculated at each week andcompared between groups by independent samples t-test. In addition,repeated-measure ANOVA was used to examine the effect of time and numberof lesions between groups.

Mean lesion size was compared similarly for weeks 0-12. For weeks 14-28,non-cancerous and cancerous lesions were categorized as ≦10 mm³versus >10 mm³ and the proportion of lesions >10 mm³ in living animalswas compared between groups by χ2-square or Fisher's exact test.Cancerous lesions were also categorized as ≦200 mm³ versus >200 mm³.

Cancer development (for weeks 14-28) and survival rates (for weeks 0-28)were evaluated using the Kaplan-Meier method and compared between groupsby log rank test. For cultured cells data, significance of differencesbetween groups was estimated by t-test, or by one-way or two-way ANOVAwith Tukey-Kramer Multiple Comparisons post test analysis.

Example XI Mouse Keratinocyte Cell Culture

The experiments utilized primary cultures of epidermal keratinocytesgenerated from 6-8 weeks old wild-type C57B1 mice (Charles River,Wilmington, Mass.); from P2X₇ ^(−/−)Pfizer mice constructed by deletionof amino acids 506-532 of the C-terminus (Solle et al., “Alteredcytokine production in mice lacking P2X(7) receptors” J Biol Chem 2001,276:125-32), or from P2X₇ ^(−/−)GSK mice, which have a lacZ geneinserted at the beginning of exon 1, resulting knockout of the receptor.Sikora et al., “Purinergic signaling regulates radical-mediatedbacterial killing mechanisms in macrophages through a P2X₇-independentmechanism” J Immunol 1999, 163:558-61. The P2X₇ ^(−/−)Pfizer and P2X₇^(−/−)GSK mice were generated on the C57B1 background.

Primary cultures of mouse keratinocytes were generated by a modifiedcollagenase-EDTA method. Li et al., “Decreased expression of P2X7 inendometrial epithelial pre-cancerous and cancer cells” Gynecol Oncology2007, 106:233-243. Ventral superficial skin areas involving the fullepidermis and part of the dermis were scraped and the cell suspensionwas washed by PBS and filtered through a 40-μm cell strainer. Followingrepeated washes and spinning cells were plated at a density of 1×10⁵cells per cm² on type-I collagen-coated filters and used for experimentsafter 24-48 hours.

P2X₇-specific antisense oligonucleotides (ASO) and random controloligonucleotides (RCO) were designed from the published sequence of themouse P2X₇ gene (Accession Number AJ009823), using a previouslydescribed method. Chessell et al., “Cloning and functionalcharacterisation of the mouse P2X₇ receptor” FEBS Lett 1998, 439:26-30;and Zhu et al., “Changes in Tight Junctional Resistance of the CervicalEpithelium are Associated with Modulation of Content and Phosphorylationof Occludin 65 KDa and 50 KDa forms” Endocrinology 2006, 147:977-989.

The sequences of the 20-mer ASO that would hybridize to the codingregion of exon 13 (nt 1467-1486), and the RCO were as follows: ASO-GGCGTA CCG CAG CAA CGT (SEQ ID NO: 1): AG; RCO-TAA GTA CTG CAG CTA CGT AC(SEQ ID NO: 2) These sequences were designed such that nocross-hybridization against the P2X₇ gene occurs. To assess the effectsof the ASO and RCO on P2X₇ mRNA expression, cultured cells were treatedfor 14 hours with or without 100 μM ASO or RCO.

Example XII Cytosolic Calcium (Ca²⁺ _(i)) and Ethidium-Bromide Assays

Changes in Ca^(2+i) in cultured cells were determined in terms ofchanges in intracellular Fluo-4 fluorescence using dynamic confocallaser scanning microscopy. Cultured mouse keratinocytes were loaded with5 μM Fluo-4/AM, and imaged with a Zeiss LSM 510 inverted real-timeconfocal microscope. Images were collected at 488 nm/505 nm (exc/emi) atintervals of 10 to 15 seconds after treatment with 100 μM BzATP, addedto the perfusate. For ethidium bromide influx experiments,glass-bottomed dishes cultured with mouse keratinocytes were placed inthe microscope. Images (collected at 488 nm/505 nm [exc/emi]) were takenbefore, and at intervals of 30 seconds after adding 5 μM ethidiumbromide to the perfusate as described. Average fluorescence intensitywas quantified from collated images using MetaVue software (FryerCompany Inc., Huntley, Ill.) by subtracting the basal intensity value.

Example XIII DNA Synthesis Assay

Changes in DNA synthesis were determined in terms of [³H]-thymidineincorporation as described Wang et al., “EGF facilitates epinephrineinhibition of P2X7-receptor mediated pore formation and apoptosis: anovel signaling network” Endocrinology 2005, 146:164-174. Theradioactivity (dpm/mg Protein, determined by Bio-Rad Protein Assaysolution [Hercules, Calif.]) of triplicated samples was determined bybeta scintillation counting (Beckman LS1801 scintillation counter).

Example IVX BzATP Effects on P2X₇ Receptor Activation

BzATP treatment was reported to augment apoptosis in vivo in mousepapilloma and in mouse cancer cells. See, FIGS. 26P, 26R and FIGS.26T,26V, respectively, as reported in [55].

The effect in papilloma cells was greater than in cancer cells. Compare,FIGS. 26R and 26V. In papilloma and in cancer cells, baseline apoptosisand the BzATP-induced augmented apoptosis correlated with receptorexpression. See, FIGS. 26P,26T and FIGS. 26R,26V, respectively. Highdensities of the receptor in papilloma keratinocytes was associated withhigh baseline apoptosis and with high degree of BzATP-augmentedapoptosis. See, FIG. 26O and FIG. 26Q. In contrast, low densities of thereceptor in cancer cells was associated with low baseline apoptosis andwith low degree of BzATP-augmented apoptosis. See, FIG. 26S and FIG.26U. Steady-state levels of ATP in extracellular fluid of normal andcancer epithelial cells are reported to be similar [38,40,42,55].Therefore, the above mouse data suggest that in vivo the degree ofreceptor activation and apoptosis is determined mainly by the level ofreceptor expression, rather than by its affinity to ATP. See, FIG. 26.

In vivo treatment of mice with BzATP resulted in 50% inhibition ofpapilloma and cancer formation. The experiments used a relatively lowdose of 1 μg/cm² BzATP, calculated based on the pre-maximalconcentration of 100 μM required for apoptosis in cultured cells[38,40-42,50,55]. The BzATP treatment involved twice weekly applicationsof the drug on the skin, with the anticipation that BzATP is absorbedfrom the application site and will reach a sufficiently highconcentration at its destined target of P2X₇-receptor-expressing reservecells. The results showed that BzATP up-regulated apoptosis ofP2X₇-receptor-expressing cells in the basal/parabasal layers and in hairshafts. However, it is unknown whether tissue levels reached theanticipated 100 μM concentration of BzATP, and it is possible that theeffect was induced by lower tissue levels of BzATP. Theoretically, itcould be possible to produce greater inhibition of skin papillomas andcancers by providing higher concentrations of BzATP at the target skinreserve cells.

In addition to drug pharmacokinetics, the data also raise the questionwhich types of keratinocytic reserve cells have responded to BzATP. Inthe normal skin treatment with BzATP induced apoptosis in most, but notin all proliferating P2X₇-receptor-expressing cells. See, FIG. 26N.

Example XV Identification of P2X₇ Gene Promoter Enhancer Sites

Cell Cultures

Primary cultures of human ectocervical-vaginal epithelial cells (hEVEC),a well-characterized model of the normal human ectocervical epithelium,were generated from discarded normal ectocervical-vaginal tissues. Wanget al., (2004) “P2X₇-receptor mediated apoptosis of human cervicalepithelial cells” Am J Physiol 287:C1349-C1358. Human cervicalepithelial cancer cell lines (Caski, Hela, Siha, and HT3), and humanembryonic kidney 293 cells (HEK293), which lack endogenous expression ofP2X₇, were obtained from the ATCC. Cell culture conditions have beendescribed. Feng et al., (2006) “A truncated P2X₇ receptor variant(P2X_(7-j)) endogenously expressed in cervical cancer cells antagonizesthe full-length P2X₇ receptor through hetero-oligomerization” J BiolChem 281:17228-17237.

Elucidation of the Promoter Region

HindIII-tailed sense primers and the NcoI-tailed antisense primers(InVitrogen, Carlsbad Calif.) were used for the elucidation of thepromoter region of the human P2X₇ gene (GenBank Y12851). See, Table 1.

TABLE 1 Primers for elucidation of the promoter region.Segments are numbered relative to theTranscription Initiation Start Site. HindIII-tailed sense −1664TTT TTA AGC TTA AGA TGT GAA GCC AGG ATC G (SEQ ID NO: 7) −1179TTT TTA AGC TTG GAT CAA GCC AGC TGTA (SEQ ID NO: 8) −698TTT TTA AGC TTG GTG GTG TCC CTC ACT GAA T (SEQ ID NO: 9) −399TTT TTA AGC TTG GGG CTG AAT AAA GGG TTG T (SEQ ID NO: 10) −204TTT TTA AGC TTA ATG CCC ATC CTC TGA ACA C (SEQ ID NO: 11) −158TTT TTA AGC TTG CCA GCT GGG GTG AGG TCA TCT G (SEQ ID NO: 12) −122TTT TTA AGC TTT AGG ACT TGG CGC TTC TTG T (SEQ ID NO: 13) −73TTT TTA AGC TTA GGG CCC GCC CCA ACT CTG GAG (SEQ ID NO: 14) −53TTT TTA AGC TTA GTC ATT GGA GGA GCT TGA AGT TA (SEQ ID NO: 15)NcoI-tailed antisense −380 TTT TTC CAT GGA CAA CCC TTT ATT CAG CCC C(SEQ ID NO: 16) +32 TTT TTC CAT GGC ACA GCA AGC CCC CTC CCA GTA(SEQ ID NO: 17) +91 TTT TTC CAT GGG GTG ACA GCC TCC CTC CCT GCG CG(SEQ ID NO: 18) +221 TTT TTC CAT GGC TTA CCA AAC GTA GGA AAA(SEQ ID NO: 19) +232 TTT TTC CAT GGC CCA GAT CCC ACT TAC CAA A(SEQ ID NO: 20) +337 TTT TTC CAT GGA GAG CAC GTC TCA GAT TCG AA(SEQ ID NO: 21) +402 TTT TTC CAT GGG CTG CAG CCT GGC ACC GTT TC(SEQ ID NO: 22) +470 TTT TTC CAT GG TGC GCG CCC TGG CGG GC(SEQ ID NO: 23) +503 TTT TTC CAT GGC CTG CGC TTT CCT ACC TTC CC(SEQ ID NO: 24) +573 TTT TTC CAT GGT CAG ATG CCA GCA TGA TCA CCA GGC(SEQ ID NO: 25)Corresponding cDNA fragments were synthesized by PCR using human genomicDNA. The PCR fragments were digested with HindIII and NcoI and ligatedinto pGL3 enhancer vector (Promega, Madison, Wis.) with HindIII and NcoIsites using Rapid DNA Ligation Kit (Roche, Indianapolis, Ind.).Reporters in this experiment and in all the experiments described belowwere confirmed by sequencing.

Control plasmids containing the pGL3 luciferase enhancer vector (5064nt) or test plasmids (P2X₇-luciferase) were transfected intosubconfluent cultured HEK293 cells that were plated 14 hours earlier in6-well plates at a density of 2.5×10⁵ cells per well. The culture mediumwas replaced with fresh medium plus 100 μl serum free medium per well,containing 2.5 μl Fugene 6 (Roche) and 750 ng DNA of the control or testvectors. For luciferase activity determinations cells were cotransfectedwith Renilla luciferase and 30 ng of the pRL-CMV vector (Promega). Zhouet al., (supra). At the completion of incubations cells were harvestedand maintained in lyses buffer for 24 hours (Promega); Firefly andRenilla luciferase activities were measured consecutively by usingDual-Luciferase Reporter Assay System (Promega), and luciferase activitywas determined in terms of Fluc/Rluc. For determinations of changes inP2X₇ and Firefly luciferase (Flue) mRNA, cells were lysed followed byRNA extraction. P2X₇ and Fluc mRNA levels were determined by Real-TimePCR (qPCR) relative to cytokeratin-18 (CK-18) or GAPDH, and expressed interms of the threshold cycle of fluorescence (Ct). Primers for P2X₇,Fluc, CK-18 and GAPDH were described. Li et al., (2006) “The P2X₇Receptor: A novel biomarker of uterine epithelial cancers” CancerEpidemiol Biomarkers Preven 15:1-8, Feng et al., (2006) “A truncatedP2X₇ receptor variant (P2X_(7-j)) endogenously expressed in cervicalcancer cells antagonizes the full-length P2X₇ receptor throughhetero-oligomerization” J Biol Chem 281:17228-17237: Li et al., (2007)“Decreased expression of P2X7 in endometrial epithelial pre-cancerousand cancer cells” Gynecol Oncology 106:233-243; Zhou et al., (2008)“Micro-RNAs miR-186 and miR-150 downregulate expression of thepro-apoptotic purinergic P2X₇ receptor by activation of instabilitysites at the 3′-untranslated region of the gene that decreasesteady-state levels of the transcript” J Biol Chem 283:28274-28286.

Elucidation of the Transcription Initiation Start Site (TpIS)

A modified 5′ Rapid Amplification of cDNA Ends method (5′-RACE) was usedwith primers (InVitrogen) as shown. See, Table 2.

TABLE 2 A. Primers for elucidation of the TranscriptionInitiation Start Site (TpIS).B. Primers for elucidation of TATA-like Sites. A Anchor Hindu-tailed dTGGA CCA AGC TTA TCG ATG TCG ACT TTT TTT TTT TTT TTT V (SEQ ID NO: 26)HindIII-including anchor GGA CCA AGC TTA TCG ATG TCG AC (SEQ ID NO: 27)Anti-sense primers Biotin-labeled (located at Exon-3)GCT CTT GGC CTT CTG TTT TG (SEQ ID NO: 28) Nested (located at Exon-2)GGT GTA GTC TGC GGT GTC AA (SEQ ID NO: 29)nested HIndIII-tailed (located at Exon-2)CCG CTA AGC TTG CTT GTC ACT CAC CAG AGC A (SEQ ID NO: 30) MutationPrimers B CATT/GTAA Forward - CAG TAC GTT TGT AAT (nt −1 to +3)TGC AGT TAC TG (SEQ ID NO: 31) Reverse - CAG TAA CTG CAA TTACAA ACG TAC TG  (SEQ ID NO: 32) TA/CC Forward - GAG CTT GAA GTC CAA(nt −31 to −30) GAC TCC TG (SEQ ID NO: 33) Reverse - CAG GAG TCT TGG ACTTCA AGC TC (SEQ ID NO: 34) AGGG/TATA Forward - GCC ACT GCC TAT ACC(nt −73 to −70) CGC CCC A (SEQ ID NO: 35) Reverse - TGG GGC GGG TAT AGGCAG TGG C (SEQ ID NO: 36) TT/CC Forward - TGG CGC TTC TTG TCC(nt −102 to −101) ATC ACA GC (SEQ ID NO: 37)Reverse - GCT GTG ATG GAC AAG AAG CGC CA (SEQ ID NO: 38)Cloning, by reverse transcription (RT), was carried out at 55° C. (totalof 20 μg RNA per reaction), using Invitrogen SuperScript™ III ReverseTranscriptase (InVitrogen). Biotin-labeled primers were used for RT toproduce cDNA, and the biotin-labeled cDNA was combined with Dynabeads inorder to concentrate the cDNA product and facilitate its purification.To this aim, the RT reaction was mixed with 40 μl Dynabeads M-280streptavidin (Invitrogen) at 25° C. for 30 min; beads were washed inbuffer containing 10 mM Tris pH 7.5, 1 mM EDTA, and 2M NaCl, and thebeads-attached cDNA was tailed using Terminal Transferase (Promega) withdATP at 37° C. for 20 min and at 70° C. for 10 min. PCR was done with aprimer pair of anchor TTT and the gene specific nested primers. A secondPCR was done with the anchor primer and the nested HindIII antisenseprimer using the first PCR products diluted 100 fold. The resulting cDNAfragment was cloned into pGL3 enhancer vector with HindIII site, andconfirmed by sequencing. The primary TpIS was identified from thesequence, beginning with adenine, and designated as site +1 (nt 1683 ofthe published human P2X7 gene sequence [GenBank Y12851]).

Oligonucleotide-directed mutagenesis of regions of interest within thepromoter region utilized the pGL3 enhancer vector reporter containingfragment −158 to +32 and the PCR method. Regions of interest andmutations were as follows: nt −1 to +3 (CATT/GTAA); nt −31 to −30(TA/CC); nt −73 to −70 (AGGG/TATA); and nt −102 to −101 (TT/CC). Primersare shown in Table 2 (supra).

Mutagenesis of CpG Sites

Mutations in the CpG sites within the 547 nt region downstream of theP2X₇ promoter were as follows: +211/+212 (CG/AA), +257/+258 (CG/TT),+278/+279 (CG/TT), +319/+320 (CG/AT), +330/+331 (CG/AT), +424/+425(CG/TT), +461/+464 (CGCG/ATTA), +453/+454 (CG/TT), +475/+476 (CG/TT),+498/+499 (CG/TT), and +548/+549 (CG/TT). using the following primers.See, Table 3.

TABLE 3 Primers for CpG Mutagenesis Experiments Segment (CpG site)Mutation Primers −158/+221 CG/AAReverse - TTT TTC CAT GGC TTA CCA AAT TTA GGA AAA G (SEQ ID NO: 39)(+211/+212) −158/+337 CG/TT Forward - ATC TCT GCA GTG GCT TAC AGC ACA(SEQ ID NO: 40) (+257/+258) CG/TTReverse - TGT GCT GTA AGC CAC TGC AGA GAT (SEQ ID NO: 41) (+278/+279)CG/AT Forward - AAG CCC GAG TTG GCA GCT TCA G (SEQ ID NO: 42)(+319/+320) CG/AT Reverse - CTG AAG CTG CCA ACT GGG GCT T(SEQ ID NO: 43) (+330/+331)Reverse - TTT TTC CAT GGA GAG CAC GTC TCA GAT TAT AAA (SEQ ID NO: 44)Reverse - TTT TTC CAT GGA GAG CAA TTC TCA GAT TCG AA (SEQ ID NO: 45)−158/+470 CG/TT Forward - CAC AGG ACA AGT TGG ATT CCT (SEQ ID NO: 46)(+424/+425) Reverse - AGG AAT CCA ACT TGT CCT GTG (SEQ ID NO: 47)(+453/+454) CG/TTReverse - Nco)TTT TTC CAT GGC CCT GCG CGC CCT GGA AGG C (SEQ ID NO: 48)(+461/+464) CGCG/ATTAReverse - TTT TCC CAT GGC CCT GTA ATC CCT GGC GGG C (SEQ ID NO: 49)−158/+573 CG/TT Forward - GCA GGG TTT GCC TGG GGA AGG TAG(SEQ ID NO: 50) (+475/+476) CG/TTReverse - CTA CCT TCC CCA GGC AAA CCC TGC (SEQ ID NO: 51) (+498/+499)CG/TT Forward - GTA GGA AAG TT AGG GCA ACA C (SEQ ID NO: 52) (+548/+549)Reverse - GTG TTG CCC TGA ACT TTC CTA C (SEQ ID NO: 53)Reverse - Nco)TTT TCC ATG GTC AGA TGC GAG CAT GAT CAC CAG GAA(SEQ ID NO:54) TGG C

cDNA fragments composed of the P2X₇ active promoter attached with thedownstream 547 nt region containing the wild-type or mutant sequenceswere inserted into a luciferase vector and transfected into HEK293cells. Promoter activity was determined in terms of luciferase activity.

P2X₇-Receptor Immunostaining

Rabbit polyclonal anti-P2X₇ receptor antibody (primary, from AlomoneLaboratories, Jerusalem, Israel) [9] and goat anti-rabbit Alexa Fluoro594 (secondary, from Invitrogen) were used in a previously describedmethod. Li et al., (2006) “The P2X₇ Receptor: A novel biomarker ofuterine epithelial cancers” Cancer Epidemiol Biomarkers Preven 15:1-8;Fu et al., (2009) “Activation of P2X₇-mediated apoptosis inhibitsDMBA/TPA-induced formation of skin papillomas and cancer in mice. BMCCancer 9:114; and Li et al., (2007) “Decreased expression of P2X7 inendometrial epithelial pre-cancerous and cancer cells” Gynecol Oncology106:233-243.

Immunofluorescence was captured in a fluorescence microscope NikonEclipse 80i (Nikon, Melville N.Y.). Image analysis of theimmunofluorescence data was performed and expressed in terms of averagepixel density per cell. Feng et al., (2006) “A truncated P2X₇ receptorvariant (P2X_(7-j)) endogenously expressed in cervical cancer cellsantagonizes the full-length P2X₇ receptor throughhetero-oligomerization” J Biol Chem 281:17228-17237.

Apoptosis Assays

Apoptosis was quantified by using Roche Cell Death Detection ELISA Kit(Roche). Feng et al., (2006) “A truncated P2X₇ receptor variant(P2X_(7-j)) endogenously expressed in cervical cancer cells antagonizesthe full-length P2X₇ receptor through hetero-oligomerization” J BiolChem 281:17228-17237.

In Vitro Methylation Assays

Control (pGL3 luciferase enhancer vector) or test plasmids(P2X₇-promoter-luciferase) were methylated in vitro prior totransfections by incubations with the CpG-Methylase M.SssI (which adds amethyl group in cytosine residues) according to the manufacturer'sprotocol (New England Biolabs, Ipswich, Mass.).

Electrophoretic Mobility Shift Assay (EMSA)

A pGL3 enhancer Vector with an insert of the P2X₇ promoter was used asthe template, and assays were performed using LightShift®Chemiluminescent EMSA Kit (Pierce, Rockford, Ill.). Briefly,biotin-labeled fragments were amplified by PCR with at least one primersas shown, See, Table 4 (includes SEQ ID NOs: 55-61 and 69).

TABLE 4 Primers for the electrophoretic mobility shift assays ForwardProduct Fragment (Biotin-labeled at 5′) Reverse Size F401-475GCAGAGAGAAGCCACAGGA GCCCTGCGCGCCCTGGCG  75 bp (SEQ ID NO: 69)(SEQ ID NO: 55) F401-530 GCAGAGAGAAGCCACAGGA CCTGCGCTTTCCTACCTTCCC103 bp (SEQ ID NO: 56) (SEQ ID NO: 57) F401-573 GCAGAGAGAAGCCACAGGATCAGATGCCATCATGATCACC 173 bp (SEQ ID NO: 58) (SEQ ID NO: 59) F217-237GTAAGTGGGATCTGGGGAGGA TCCTCCCCAGATCCCACTTAC  21 bp (SEQ ID NO: 60)(SEQ ID NO: 61)

After amplification, P2X₇ promoter fragments were separated on 1.5%agarose gel and extracted using the Agarose Gel Extraction Kit (QIAGEN).The binding reaction was performed for 30 min at room temperature usingthe EMSA kit system in a total volume of 20 μL containing 2 μl bindingbuffer, 0.5 μg of HeLaScribe Nuclear Extract (Promega), 40 fmolbiotin-labeled oligonucleotide, and 1 μl poly (dI-dC). DNA-proteincomplexes formed were fractionated by electrophoresis over 4%polyacrylamide gels in 1× Tris-boric acid-EDTA buffer. Gels wereelectrophoretically transferred at 100V for 1 hour on ice to apositively charged nylon membrane and immediately cross-linked with a UVtransilluminator. Streptavidin-horseradish peroxidase conjugate and theLightShift Chemiluminescent Substrate (Pierce) were used to detect thebiotin end-labeled DNA, and the nylon membranes were exposed to x-rayfilm for 3 minutes for detection of possible DNA-protein bindingreactions.

Human Tissues

Discarded human uterine tissues were used for DNA methylation analysis.Tissues were obtained from the Human Tissue Procurement Facility ofUniversity Hospital CASE Medical Center, Case Western ReserveUniversity, Cleveland Ohio, according to approved research protocols12-03-50 and 03-90-300 by the Institutional Review Board. Cross sectionsof cervical segments were obtained from paraffin embedded blocks thatwere prepared by the Department of Pathology initially to establish thepatient's diagnosis. For the assays, additional parallel 10 μm sectionswere cut and slides were made according to standard procedures. For eachcase an H&E stained slide was used to identify normal or cancerouscervical epithelial regions. Tissue epithelial fragments were obtainedfrom parallel regions of non-stained slides by microdissection. Thetissue material was dispersed into 100 μl digestion buffer (50 mM TrispH 8.5, 0.5% Tween 20, 200 μg/ml proteinase-K), incubated at 42° C.overnight, and proteinase-K was inactivated by incubation at 80° C. for10 min.

DNA Methylation Analysis

Assays used the method of combined bisulfite restriction analysis(COBRA) which employs restriction enzyme digestion to revealmethylation-dependent sequence differences in PCR products of sodiumbisulfite-treated DNA. Xiong et al., (1997) “COBRA: a sensitive andquantitative DNA methylation assay” Nucleic Acid Research 25:2532-2534.Briefly, bisulfite-treated, genomic DNA unmethylated cytosines areconverted to thymidines whereas methylated cytosine residuals areretained as cytosines. DNA segments of interest are amplified using PCRprimers that do not contain CpG dinucleotides so that the amplificationstep does not discriminate between templates by their originalmethylation status. The PCR products are digested by restriction enzymesthat recognize sequences containing CpG. Cleavage occurs if the CpGsequence has been retained during the bisulfite conversion according tothe methylated status of the cytosine residue. The digested PCR productsare resolved and separated by gel electrophoresis and stained withethidium bromide.

Cloning of CpG-Rich Regions Downstream of the Promoter

Patterns of methylation were determined within a 547 by region of nt+26/+573 relative to a TpIS, nt +1. See, FIG. 28. The region of interestwas arbitrarily divided to three segments with partial overlaps,designated Segment-1 (nt +26/+247), Segment-2 (nt +223/+399), andSegment-3 (nt +352/+573) wherein the following primers were used. See,Table 5.

TABLE 5 Primers for experiments using Segments 1-3 Segment-1 Forward -TGT TGT GGT TTT GTT AGG AAG AGT A (SEQ ID NO: 63) Reverse -AAA AAT CTA AAT CCT CCC CAA ATC (SEQ ID NO: 64) Segment-2 Forward -GGA TTT GGG GAG GAT TTAGATT-3 (SEQ ID NO: 65) Reverse -CAA CCT AAC ACC GTT TCCTCTT-3 (SEQ ID NO: 66) Segment-3 Forward -GGG AGG GAG GAA GTA GTA GTA GGT A (SEQ ID NO: 67) Reverse -TCA AAT ACC AAC ATA ATC ACC AAA C (SEQ ID NO: 68)PCR conditions (annealing temperature) and the restriction enzyme usedwere as follows: Segment-1 560C (MaeII); Segment-2 560C (MaeII);Segment-3 590C (BstUI).Evaluation of DNA Methylation

Genomic DNA was extracted from cultured cells and from human tissuesusing a DNA purification kit (Promega). To convert CpG non-associatedcytosines to uracil (and thymidines), 1 mg of genomic DNA was denaturedwith 2 M NaOH at 37° C. for 10 min; 30 μl of 10 mM hydroquinone wereadded and the solution was incubated with 3M sodium bisulfite (pH 5) at53° C. for 16 hours in darkness. After treatment, DNA was purified byDNA clean up kit (Promega). The solution was incubated with 2 M NaOH atroom temperature and precipitated with 100% ethanol, washed with 70%ethanol, and resuspended in 20 μl of distilled water. The bisulfitetreated DNA was amplified by PCR (95° C. 5 min/95° C. 30 sec/55-59° C.45 sec/and 72° C. 45 sec, 37 cycles). PCR products were digested withrestriction enzymes and separated in 6% polyacrylamide gel at 4 W for 3hours and the amplified products were checked for accuracy bysequencing. Positive control for the DNA methylation experiments washuman placental genomic DNA treated with the CpG-Methylase M.SssI andthe negative control was water. DNA methylation was determined from thepolyacrylamide gel pictures in terms of samples showing de-novoappearance of a low MW band, corresponding to a cleaved band at apreviously methylated site. The degree of methylation was determined bydensitometry in terms of the intensity of the cleaved fragment (lower MWband) relative to the density of the non-methylated plus the methylatedbands (lower plus higher MW bands). Densitometry was done as described.Feng et al., (2006) “A truncated P2X₇ receptor variant (P2X_(7-j))endogenously expressed in cervical cancer cells antagonizes thefull-length P2X₇ receptor through hetero-oligomerization” J Biol Chem281:17228-17237.

To confirm the efficacy of the bisulfate method for converting genomicDNA cytosines (but not methylcytosines) to uracils, bisulfate-treatedgenomic DNA was amplified with primers for Segments 1 and 2. PCRproducts were purified with PCR purification kit (QIAGEN) and clonedinto vector pCR II-TOPO (Invitrogen). Subclones were transformed intoE-Coli bacteria type BL21 and cultured overnight at 37° C.Plasmid-containing PCR products were extracted with miniprep kit(Promega) and sequenced. The results (not shown) confirmed that allgenomic DNA cytosines converted to uracils, except methylcytosines.

Data Analysis

Data were analyzed using GraphPad Instat (GraphPad Software Inc.,San-Diego, Calif.). Significance of differences between groups wasestimated by t-test, or by one-way or two-way ANOVA with Tukey-KramerMultiple Comparisons post test analysis.

Supplies

All chemicals, unless specified otherwise, were obtained from SigmaChemicals (St. Louis, Mo.).

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1. A method for inducing apoptosis in malignant cancer cells,comprising: a) providing: i) a subject comprising a plurality ofmalignant cancer cells, wherein said cancer cells comprise a P2X₇receptor; ii) a composition comprising a compound that pharmacologicallyactivates P2X₇ mediated apoptosis in said cancer cells; b) administeringsaid composition locally to said cancer cells under conditions such thatapoptosis is induced.
 2. The method of claim 1, wherein said compoundcomprises benzoylbenzoyl adenosine triphosphate (BzATP).
 3. The methodof claim 1, wherein said apoptosis kills said cancer cell.
 4. The methodof claim 1, wherein said local administering comprises using a medicaldevice selected from the group consisting of a transdermal patch, acatheter, an applicator gun, and a syringe.
 5. The method of claim 1,wherein said composition further comprises TNFα.
 6. The method of claim1, wherein said plurality of cancer cells are derived from epithelialcells.
 7. The method of claim 1, wherein said plurality of cancer cellsare selected from the group consisting of epidermal cancer cells andsquamous cell carcinoma cells.
 8. A method for inducing apoptosis inmalignant epithelial cancer cells, comprising: a) providing: i) asubject comprising a plurality of malignant epithelial cancer cells; ii)a benzoylbenzoyl-adenosine triphosphate P2X₇ receptor agonist capable ofinducing apoptosis in said cancer cells; b) administering said agonistto the surface of said cancer cells under conditions such that apoptosisis induced.
 9. The method of claim 8, wherein said apoptosis kills saidcancer cell.
 10. The method of claim 8, wherein said administeringfurther comprises a medical device.
 11. The method of claim 10, whereinsaid medical device is selected from the group consisting of atransdermal patch, a catheter, an applicator gun and a syringe.
 12. Themethod of claim 8, wherein said administering further comprises TNFα.13. The method of claim 8, wherein said plurality of epithelial cancercells are germative epithelial cancer cells.
 14. The method of claim 8,wherein said plurality of epithelial cancer cells are selected from thegroup consisting of epidermal cancer cells and squamous cell carcinomacells.